Family of immunoregulators designated leukocyte immunoglobulin-like receptors (LIR)

ABSTRACT

A new family of immunoreceptor molecules of the immunoglobulin superfamily, (LIR) polypeptides is described. Disclosed are sequences encoding LIR family members and their deduced amino acid sequences, polypeptides encoded by DNA that hybridize to oligonucleotide probes having defined sequences, processes for producing polypeptides of the LIR family, and antagonistic antibodies to LIR family members. LIR family members can be used to treat autoimmune diseases and disease states associated with suppressed immune function.

BACKGROUND OF THE INVENTION

[0001] Immune system cellular activity is controlled by a complexnetwork of cell surface interactions and associated signaling processes.When a cell surface receptor is activated by its ligand a signal is sentto the cell, and, depending upon the signal transduction pathway that isengaged, the signal can be inhibitory or activatory. For many receptorsystems cellular activity is regulated by a balance between activatorysignals and inhibitory signals. In some of these it is known thatpositive signals associated with the engagement of a cell surfacereceptor by its ligand are downmodulated or inhibited by negativesignals sent by the engagement of a different cell surface receptor byits ligand.

[0002] The biochemical mechanisms of these positive and negativesignaling pathways have been studied for a number of known immune systemreceptor and ligand interactions. Many receptors that mediate positivesignaling have cytoplasmic tails containing sites of tyrosinephosphatase phosphorylation known as immunoreceptor tyrosine-basedactivation motifs (ITAM). A common mechanistic pathway for positivesignaling involves the activation of tyrosine kinases whichphosphorylate sites on the cytoplasmic domains of the receptors and onother signaling molecules. Once the receptors are phosphorylated,binding sites for signal transduction molecules are created whichinitiate the signaling pathways and activate the cell. The inhibitorypathways involve receptors having immunoreceptor tyrosine basedinhibitory motifs (ITIM), which, like the lTAMs, are phosphorylated bytyrosine kinases. Receptors having these motifs are involved ininhibitory signaling because these motifs provide binding sites fortyrosine phosphatases which block signaling by removing tyrosine fromactivated receptors or signal transduction molecules. While many of thedetails of the activation and inhibitory mechanisms are unknown, it isclear that functional balance in the immune system depends upon opposingactivatory and inhibitory signals.

[0003] One example of immune system activity that is regulated by abalance of positive and negative signaling is B cell proliferation. TheB cell antigen receptor is a B cell surface immunoglobulin which, whenbound to antigen, mediates a positive signal leading to B cellproliferation. However, B cells also express Fcγ RIIb1, a low affinityIgG receptor. When an antigen is part of an immune complex with solubleimmunoglobulin, the immune complex can bind B cells by engaging both theB cell antigen receptor via the antigen and Fcγ RIIb1 via the solubleimmunoglobulin. Co-engagement of the Fcγ RIIb1 with the B cell receptorcomplex downmodulates the activation signal and prevents B cellproliferation. Fcγ RIIb1 receptors contain ITIM motifs which are thoughtto deliver inhibitory signals to B cells via interaction of the ITIMswith tyrosine phosphatases upon co-engagement with B cell receptors.

[0004] The cytolytic activity of Natural Killer (NK) cells is anotherexample of immune system activity which is regulated by a balancebetween positive signals that initiate cell function and inhibitorysignals which prevent the activity. The receptors that activate NKcytotoxic activity are not fully understood. However, if the targetcells express cell-surface MHC class I antigens for which the NK cellhas a specific receptor, the target cell is protected from NK killing.These specific receptors, known as Killer Inhibitory Receptors (KIRs)send a negative signal when engaged by their MHC ligand, downregulatingNK cell cytotoxic activity.

[0005] KIRs belong to the immunoglobulin superfamily or the C-typelectin family (see Lanier et al., Immunology Today 17:86-91,1996). Knownhuman NK KIRs are members of the immunoglobulin superfamily and displaydifferences and similarities in their extracellular, transmembrane andcytoplasmic regions. A cytoplasmic domain amino acid sequence common tomany of the KIRs is an ITIM motif having the sequence YxxL/V. In somecases, it has been shown that phosphorylated ITIMs recruit tyrosinephosphatases which dephosphorylate molecules in the signal transductionpathway and prevent cell activation (see Burshtyn et al., Immunity4:77-85, 1996). The KIRs commonly have two of these motifs spaced apartby 26 amino acids [YxxL/V(x)₂₆YxxL/V]. At least two NK cell receptors,each specific for a human leukocyte antigen (HLA) C allele (an MHC classI molecule), exist as an inhibitory and an activatory receptor. Thesereceptors are highly homologous in the extracellular portions, but havemajor differences in their transmembrane and cytoplasmic portions. Oneof the differences is the appearance of the ITIM motif in the inhibitoryreceptor and the lack of the ITIM motif in the activating receptor (seeBiassoni et al., Journal. Exp. Med, 183:645-650, 1996).

[0006] An immunoreceptor expressed by mouse mast cells, gp49B1, also amember of the immunoglobulin superfamily, is known to downregulate cellactivation signals and contains a pair of ITIM motifs. gp49B1 shares ahigh degree of homology with human KIRs (Katz et al., Cell Biology, 93:10809-10814, 1996). Mouse NK cells also express a family ofimmunoreceptors, the Ly49 family, which contain the ITIM motif andfunction in a manner similar to human KIRs. However, the Ly49immunoreceptors have no structural homology with human KIRs and containan extracellular C-type lectin domain, making them a member of thelectin superfamily of molecules (see Lanier et al., Immunology Today17:86-91, 1996).

[0007] Clearly, the immune system activatory and inhibitory signalsmediated by opposing kinases and phosphatases are very important formaintaining balance in the immune system. Systems with a predominance ofactivatory signals will lead to autoimmunity and inflammation. Immunesystems with a predominance of inhibitory signals are less able tochallenge infected cells or cancer cells. Isolating new activatory orinhibitory receptors is highly desirable for studying the biologicalsignal(s) transduced via the receptor. Additionally, identifying suchmolecules provides a means of regulating and treating diseased statesassociated with autoimmunity, inflammation and infection.

[0008] For example engaging a newly discovered cell surface receptorhaving ITIM motifs with an agonistic antibody or ligand can be used todownregulate a cell function in disease states in which the immunesystem is overactive and excessive inflammation or immunopathology ispresent. On the other hand, using an antagonistic antibody specific tothe receptor or a soluble form of the receptor can be used to block theinteraction of the cell surface receptor with the receptor's ligand toactivate the specific immune function in disease states associated withsuppressed immune function. Conversely, since receptors lacking the ITIMmotif send activatory signals once engaged as described above, theeffect of antibodies and soluble receptors is the opposite of that justdescribed.

SUMMARY OF THE INVENTION

[0009] The present invention provides a new family of immunoreceptormolecules of the immunoglobulin superfamily, designated herein as theLeukocyte Immunoglobulin-Like Receptor (LIR) polypeptides. Within thescope of the present invention are DNA sequences encoding LIR familymembers and their deduced amino acid sequences disclosed herein. Furtherincluded in the present invention are polypeptides encoded by DNA thathybridize to oligonucleotide probes having defined sequences or to DNAor RNA complementary to the probes. The present invention also includesrecombinant expression vectors comprising DNA encoding LIR familymembers. Also within the scope of the present invention are nucleotidesequences which, due to the degeneracy of the genetic code, encodepolypeptides substantially identical or substantially similar topolypeptides encoded by the nucleic acid sequences described above, andsequences complementary to those nucleotide sequences.

[0010] Further, the present invention includes processes for producingpolypeptides of the LIR family by culturing host cells transformed witha recombinant expression vector that contains an LIR family memberencoding DNA sequence under conditions appropriate for expressing an LIRpolypeptide family member, then recovering the expressed LIR polypeptidefrom the culture. The invention also provides agonistic and antagonisticantibodies to LIR and LIR family members.

[0011] Further still within the present invention are fusion proteinswhich include a soluble portion of an LIR family member and the Fcportion of Ig.

[0012] Disorders mediated by autoimmune disease associated with failureof a negative signaling LIR to downregulate cell function may be treatedby administering a therapeutically effective amount of an agonisticantibody or ligand of LIR family members to a patient afflicted withsuch a disorder. Disorders mediated by disease states associated withsuppressed immune function can be treated by administering a solubleform of the negative signaling LIR. Conversely, disorders mediated bydiseases associated with failure of a activatory signaling LIR can betreated by administering an agonistic antibody of the activatoryreceptor. Disorders mediated by states associated with autoimmunefunction can be treated by administering a soluble form of theactivatory receptor.

DETAILED DESCRIPTION OF THE INVENTION

[0013] A viral glycoprotein having a sequence similarity to MHC class Iantigens has been used to isolate and identify a new polypeptide,designated LIR-P3G2, and a new family of cell surface polypeptidesdesignated the LIR polypeptide family. The LIR polypeptide familymembers possess extracellular regions having immunoglobulin-likedomains, placing the members in a new subfamily of the immunoglobulinsuperfamily. While, the LIR family members are characterized as havingvery similar extracellular portions, the family includes three groups ofpolypeptides which are distinguishable by their transmembrane regionsand their cytoplasmic regions. One group of the LIR polypeptides has atransmembrane region that includes a positively charged residue and ashort cytoplasmic tail and a second group has a nonpolar transmembraneregion and a long cytoplasmic tail. A third group includes a polypeptideexpressed as a soluble protein having no transmembrane region orcytoplasmic tail. LIR-P3G2 is expressed by a variety of cells andrecognizes HLA-B44 and HLA-A2 MHC molecules and, by analogy with knownmolecules, LIR-P3G2 has a role in immune recognition and self/nonselfdiscrimination.

[0014] Examples 1-3 below describe isolating cDNA encoding P3G2(LIR-P3G2) and a substantially identical polypeptide designated 18A3(LIR-18A3). Briefly, the LIR-P3G2 family member was isolated by furstexpressing UL18, a Class I MHC-like molecule and using UL18 to isolateand identify P3G2 and 18A3. The nucleotide sequences of the isolatedP3G2 cDNA and 18A3 cDNA are presented in SEQ ID NO:1 and SEQ ID NO:3,respectively. The amino acid sequences encoded by the cDNA presented inSEQ ID NO:1 and SEQ ID NO:3 are presented in SEQ ID NO:2 and SEQ IDNO:4, respectively. The P3G2 amino acid sequence (SEQ ID NO:2) has apredicted extracellular domain of 458 amino acids (1-458) including asignal peptide of 16 amino acids (amino acids 1-16); a transmembranedomain of 25 amino acids (amino acids 459-483) and, a cytoplasmic domainof 167 amino acids (amino acids 484-650). The extracellular domainincludes four immunoglobulin-like domains. Ig-like domain I includesapproximately amino acids 17-118; Ig-like domain II includesapproximately amino acids 119-220; Ig-like domain III includesapproximately amino acids 221-318; and Ig-like domain IV includesapproximately amino acids 319-419. Significantly, the cytoplasmic domainof this polypeptide includes four ITIM motifs, each having the consensussequence of YxxL/V. The first ITIM motif pair is found at amino acids533-536 and 562-565 and the second pair is found at amino acids 614-617and 644-647. This feature is identical to the ITIM motifs found in KIRsexcept that KIRs contain only one pair of ITIM motifs.

[0015] The 18A3 amino acid sequence has a predicted extracellular regionof 459 amino acids (1-459) including a signal peptide of 16 amino acids(amino acids 1-16); a transmembrane domain of 25 amino acids (aminoacids 460-484) and a cytoplasmic domain of 168 amino acids (485-652).The 18A3 amino acids sequence (SEQ ID NO:4) is substantially identicalto that of P3G2 (SEQ ID NO:2) except that 18A3 has two additional aminoacids (at amino acid 438 and 552) and 18A3 possesses an isoleucineresidue at amino acid 142 in contrast to a threonine residue for P3G2.Additionally, 18A3 has a serine residue at amino acid 155 and P3G2 hasan isoleucine at 155. Finally, the 18A3 polypeptide has a glutamic acidat amino acid 627 and P3G2 has a lysine at 625 which is aligned with the627 residue of the 18A3 polypeptide. ITIM motifs in the 18A3 cytoplasmicdomain are at amino acids 534-537 and 564-567 and at 616-619 and646-649. Glycosylation sites occur at the amino acid triplet Asn-X-Y,where X is any amino acid except Pro and Y is Ser or Thr. Thus,potential glycosylation sites on LIR-P3G2 occur at amino acids 140-142;281-283; 302-304; and 341-343. Sites on LIR-18A3 are at 281-283;302-304; and 341-343. The features of these encoded polypeptides areconsistent with type I transmembrane glycoproteins.

[0016] Example 8-10 describe isolating and identifying eight additionalLIR polypeptide family members by probing cDNA libraries for plasmidsthat hybridize to a probe obtained from DNA encoding the extracellularregion of LIR-P3G2. The nucleotide sequences (cDNA) of the isolated LIRfamily members are presented in SEQ ID NO:7 (designated pbm25), SEQ IDNO:9 (designated pbm8), SEQ ID NO:11 (designated pbm36-2), SEQ ID NO:13(designated pbm36-4); SEQ ID NO:15 (designated pbmhh); SEQ ID NO:17(designated pbm2), SEQ ID NO:19 (designated pbm17) and SEQ ID NO:21(designated pbmnew). The amino acid sequences encoded thereby arepresented in SEQ ID NO:8 (designated pbm25), SEQ ID NO: 10 (designatedpbm8), SEQ ID NO:12 (designated pbm36-2), SEQ ID NO:14 (designatedpbm36-4), SEQ ID NO: 16 (designated pbmhh); SEQ ID NO: 18 (designatedpbm2); SEQ ID NO: 20 (designated pbm17), and SEQ ID NO:22 (designatedpbmnew), respectively.

[0017] The identified extracellular, transmembrane and cytoplasmicregions for the LIR family members of SEQ ID NO:10, 12, 14, 16, 18, 20,and 22 are presented below. The polypeptide presented in SEQ ID NO:8 isa soluble protein having no transmembrane and cytoplasmic regions. Aswill be understood by the skilled artisan, the transmembrane region ofP3G2 and 18A3 described above and those of LIR polypeptide familymembers presented below are identified in accordance with conventionalcriteria for identifying hydrophobic domains associated with suchregions. Accordingly, the precise boundaries of any selectedtransmembrane region may vary from those presented above. Typically, thetransmembrane domain does not vary by more than five amino acids oneither end of the domain. Computer programs known in the art and usefulfor identifying such hydrophobic regions in proteins are available.

[0018] The polypeptide presented in SEQ ID NO:8 (LIR-pbm25) has a anextracellular domain that includes the entire amino acid sequence ofamino acids 1-439 and a signal peptide of amino acids 1-16. The aminoacid sequence presented in SEQ ID NO: 10 (LIR-pbm8) has a predictedextracellular region of 458 amino acids (1458) including a 16 amino acidsignal peptide (amino acids 1-16); a transmembrane domain that includesamino acids 459-483; and a cytoplasmic domain that includes amino acids484-598. The extracellular domain includes four immunoglobulin-likedomains and the cytoplasmic domain includes an ITIM motif at amino acids533-536 and 562-565.

[0019] The amino acid sequence presented in SEQ ID NO:12 (LIR-pbm36-2)has a predicted extracellular domain of amino acids including a 16 aminoacid signal peptide of from amino acids 1-16; a transmembrane domainwhich includes amino acids 262-280 and a cytoplasmic domain of fromamino acids 281-289. The transmembrane domain includes a chargedarginine residue at 264 and the cytoplasmic domain is short, having onlya length of only 9 amino acids.

[0020] The amino acid sequence presented in SEQ ID NO:14 (LIR-pbm36-4)has a predicted extracellular domain of amino acids 1-461 including asignal peptide from amino acids 1-16; a transmembrane domain thatincludes amino acids 462-480 and possesses a charged arginine residue atamino acid 464; and a cytoplasmic domain that includes amino acids481-489. SEQ ID NO:14 is nearly identical to that of SEQ ID NO:12 exceptthat it possesses four immunoglobulin domains in contrast to the twodomains found in the extracellular region SEQ ID NO:12. The amino acidsequences presented in SEQ ID NO:12 and SEQ ID NO:14 are likely proteinsencoded by alternatively spliced transcripts from the same gene.

[0021] The amino acid sequence presented in SEQ ID NO:16 (LIR-pbmhh) hasa predicted extracellular domain that includes amino acids 1-449 and asignal peptide from amino acids 1-16; a transmembrane domain thatincludes amino acids 450-468 with a charged arginine residue at aminoacid 452; and a cytoplasmic domain that includes amino acids 469-483.The cytoplasmic domain is short with a length of 15 amino acids. Theextracellular domain includes four immunoglobulin-like domains.

[0022] The amino acid sequence presented in SEQ ID NO:18 (LIR-pbm2) hasa predicted extracellular region that includes amino acids 1-259 and asignal peptide of amino acids 1-16; a transmembrane domain that includesamino acids 260-280; and a cytoplasmic domain that includes amino acids281-448. This LIR family member has cytoplasmic domain which includes anITIM motif at amino acids 412-415 and 442-445. The extracellular domainincludes two immunoglobulin-like domains.

[0023] The amino acid sequence presented in SEQ ID NO:20 (LIR-pbm17) hasa predicted extracellular domain of amino acids 1-443 that includes asignal peptide of amino acids 1-16; a transmembrane domain whichincludes amino acids 444-464; and a cytoplasmic domain of amino acids465-631. The extracellular domain has four immunoglobulin-like domains.SEQ ID NO:20 has two pairs of ITIM YxxL/V motifs in the cytoplasmicdomain. A first pair is at amino acids 514-517 and 543-546, and a secondpair is at amino acids 595-598 and 625-628.

[0024] The amino acid sequence presented in SEQ ID NO:22 (LIR-pbmnew)has a predicted extracellular domain of amino acids 1-456 including asignal peptide of amino acids 1-16; a transmembrane domain whichincludes amino acids 457-579; and a cytoplasmic domain of amino acids580-590. The extracellular includes four immunoglobulin-like domains.SEQ ID NO:22 has an ITIM motif at amino acid 554-557 and 584-587.

[0025] The sequences presented in SEQ ID NO: 2, 4, 8, 10, 12, 14, 16,18, 20, and 22 reveal that the LIR family includes three groups ofpolypeptides. One group includes the polypeptides of SEQ ID NO: 12, 14and 16 which are distinguishable by a charged arginine residue in theirtransmembrane regions and their short cytoplasmic regions. A secondgroup includes SEQ ID NO: 2, 4, 10, 18, 20 and 22 which aredistinguishable by the hydrophobic cytoplasmic domains and the presenceof the ITIM motif in their cytoplasmic regions. The third group includesthe polypeptide of SEQ ID NO: 8 which is expressed as a solublepolypeptide and has no transmembrane region. This soluble polypeptidemay function to block the interactions of cell surface family memberswith their receptors. Alternatively, this soluble polypeptide may act asan activatory signal when it binds to its receptor. The LIR polypeptidesare characterized generally by the ability of their encoding DNA tohybridize to DNA encoding the P3G2 extracellular region.

[0026] The extracellular regions of the LIR family members presented inSEQ ID NO:2, 4, 8, 10, 12, 14, 16, 18, 20, and 22 have a high degree ofhomology which varies from 59%-84%. The extracellular regions of SEQ IDNO: 12 and SEQ ID NO:14 share sequence homology which is close to 100%since these polypeptides are from the same gene. Similarly, SEQ ID NO:2and SEQ ID NO:4 share sequence homology that is in excess of 95% and itis thought that these may be alleles of the same gene. While sharingsome structural similarities with other members of the immunoglobulinsuperfamily, the LIR family members have limited homology to thesemembers of the immunoglobulin superfamily. Molecules having the closeststructural similarity are the human KIRs and mouse gp49. However, LIRextracellular regions share only a 38-42% identity with theextracellular regions of NKAT3 and p58 Cl-39, respectively. Theextracellular regions of the LIR family members are only 35-47%homologous with that of mouse gp49. In contrast, KIRs in general areknown to share at least a 80% amino acid identity, with NKAT3 and p58CL-39 being 81% homologous. Additionally, none of the known KIRmolecules has four extracellular immunoglobulin domains which ischaracteristic of all but two of the known LIR family members. In viewof the high sequence homology among the LIR related polypeptidesdisclosed herein and their relatively low homology with KIRs, the LIRpolypeptides are members of a new family of immunoregulators.

[0027] An analysis of the amino acid sequences of the LIR polypeptidesreveals that specific stretches of amino acids of the LIR polypeptidesare highly conserved. One conserved region is the sequence of aminoacids 5-50. A data base search determined that the LIR family membersdiffer substantially from the most structurally similar prior artpolypeptides in this LIR conserved region. The data base search andstructural analysis was performed using BLAST NB1, a local alignmentsearch tool for searching data bases and aligning amino acid sequencesto determine identities and variations in a given sequence. The BLASTNB1 software is accessible on the internet athttp://www3.ncb1.nlm.nih.gov/entrez/blast. The BLAST NB1 search forsequences having homology to the sequence of amino acids 5 to 50 of SEQID NO:2 found that the most structurally similar proteins are FcγIIR,gp49B form 2, and gp49B form 1 having identities with amino acids 5 to50 of SEQ ID NO:2 of 63%, 67%, and 67% respectively. This contrasts withan LIR family identity with amino acids 5 to 50 of SEQ ID NO:2 whichranges from 77% to 100%. Specifically, LIR family members of the presentinvention have the following identities with amino acids 5-50 of SEQ IDNO:2: SEQ ID NO: 8 has a 96%; SEQ ID NO:10 has a 90% identity; SEQ IDNO:12 has a 96% identity; SEQ ID NO:14 has a 91% identity; SEQ ID NO:16has a 97% identity; SEQ ID NO:18 has a 77% identity; SEQ ID NO:20 has an80% identity; and, SEQ ID NO:22 has an 80% identity.

[0028] Sequence identity as used herein is the number of aligned aminoacids which are identical, divided by the total number of amino acids inthe shorter of the two sequences being compared. A number of computerprograms are available commercially for aligning sequences anddetermining sequence identities and variations. These programs provideidentity information based upon the above stated definition of identity.One suitable computer program is the GAP program, version 6.0, describedby Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available fromthe University of Wisconsin Genetics Computer Group (UWGCG). The GAPprogram utilizes the alignment method of Needleman and Wunsch (J. Mol.Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math2:482, 1981). The preferred default parameters for the GAP programinclude: (1) a unary comparison matrix (containing a value of 1 foridentities and 0 for non-identities) for nucleotides, and the weightedcomparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745,1986, as described by Schwartz and Dayhoff, eds., Atlas of ProteinSequence and Structure, National Biomedical Research Foundation, pp.353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10penalty for each symbol in each gap; and (3) no penalty for end gaps.Another similar program, also available from the University of Wisconsinas part of the GCG computer package for sequence manipulation is theBESTFIT program.

[0029] In another aspect, the polypeptides of the present invention haveconserved regions which are uniquely characterized as having the aminoacid sequence (SEQ ID NO:28):

[0030] Leu Xaa_(a) Leu Ser Xaa_(b) Xaa_(c) Pro Arg Thr Xaa_(d) Xaa_(e)Gln Xaa_(f) Gly Xaa_(g) Xaa_(h) Pro Xaa_(i) Pro Thr Leu Trp Ala Glu ProXaa_(j) Ser Phe Ile Xaa_(j) Xaa₇₀ Ser Asp Pro Lys Leu Xaa_(k) Leu ValXaa_(m) Thr Gly

[0031] where Xaa_(a) is Gly or Arg; Xaa_(b) is Leu or Val; Xaa_(c) isGly or Asp; Xaa_(d) is His Arg or Cys; Xaa_(e) is Val or Met; Xaa_(f) isAla or Thr, Xaa_(g) is His Pro or Thr, Xaa_(h) Leu Ile or Phe; Xaa_(i)is Gly Asp or Ala; Xaa_(j) is Thr Ile Ser or Ala; Xaa_(k) is Gly or Val;Xaa_(m) is Met or Ala; and Xaa₇₀ is a sequence of 70 amino acids.

[0032] As mentioned above, certain LIR family have ITIM motifs(YxxL/V₂₅₋₂₆YxxL/V) in their cytoplasmic domains. It is known that manyimmune regulating receptors such as KIRs, CD22, FcγRIIb1 also have ITIMsin their cytoplasmic domain and function to send inhibitory signalswhich down regulate or inhibit cell function. It has been shown thatthese receptors associate with SHP-1 phosphatase via binding to the ITIMmotifs. Recruitment of the SHP-1 phosphatase by the receptor appears tobe required for intracellular signaling pathways that regulate theinhibitory function of the receptors. The experiment described inExample 11 demonstrates that LIR-P3G2 associates with SHP-1 phosphatase.It is known that many immune regulating receptors such as KIRs, CD22,FcγRIIb1 have ITIMs in their cytoplasmic domain and function to sendinhibitory signals which down regulate or inhibit cell function. Thus,by analogy with KIRs, CD22 and FcγRIIb1, LIR family members presented inSEQ ID NO:2, 4, 10, 18, 20, and 22 that have ITIM motifs, deliver aninhibitory signal via the interaction of its ITIM with SHP-1 tyrosinephosphatase, or other tyrosine phosphatases, when the LIR is coligatedwith an appropriate receptor. Also by analogy with immunoregulatoryreceptors possessing ITIMs, LIR family members have a regulatoryinfluence on humoral, inflammatory and allergic responses.

[0033] The LIR family members presented in SEQ ID NO:12, 14, and 16 haverelatively short cytoplasmic domains, have transmembrane regionspossessing at least one charged residue, and do not possess the ITIMmotif. By analogy with membrane proteins that lack ITIM motifs and havecharged transmembrane regions, these family members mediate stimulatoryor activatory signals to cells. For example, membrane bound proteinscontaining a charged residue in the transmembrane regions are known toassociate with other membrane-bound proteins that possess cytoplasmictails having motifs known as immunoreceptor tyrosine-based activationmotifs (ITAM). Upon association, the ITAMs become phosphorylated andpropagate an activation signal.

[0034] The LIR polypeptide designated LIR-P3G2 is expressed on thesurface of transfected or normal cells. This is evidenced by the resultsof the experiments described in Example 3 and Example 5 in which flowcytometry and precipitation techniques demonstrate that LIR-P3G2 isfound on monocytes, a subpopulation of NK cells, and B cells. P3G2 wasnot detected on T cells. P3G2 is expressed as a 110-120 kDaglycoprotein. Since P3G2 has four potential glycosylation sites, themolecular size will vary with the degree of its glycosylation.Glycosylation sites occur at the amino acid triplet Asn-X-Y, where X isany amino acid except Pro and Y is Ser or Thr. Potential glycosylationsites on P3G2 occur at amino acids 139-141; 280-282; 302-304; and340-342.

[0035] P3G2-LIR isolated as described in Example 3 was tested for itsability to bind to cell surface ligands distinct from UL18. Asdemonstrated by the experimental results detailed in Example 7, P3G2binds HLA-B 44 and HLA-A2, class I MHC antigens. Since Class I MHCmolecules play a central role in immune surveillance, self/non-selfdiscrimination, the immune response to infection etc., the LIR-P3G2polypeptide has a role in regulation of immune responses. It is knownthat NK cytolytic activity for killing tumor cells and cells infectedwith a virus is regulated by a delicate modulation of activatory andinhibitory signals. It has been shown that receptors specific for thesame HLA class I molecules to which P3G2 binds may be activatory orinhibitory in their triggering mechanism. By analogy, P3G2 which bindsMHC class I molecules, plays a role in balancing immune system cellactivity and is useful in treating disease states in which the immunesystem balance is disrupted.

[0036] Within the scope of the present invention are polypeptides whichinclude amino acid sequences encoded by DNA that hybridizes to LIR-P3G2extracellular DNA probes under moderate to highly stringent conditionsas taught herein. Probes which hybridize to DNA that encode polypeptidesof the present invention include probes which encompass nucleotides310-1684 of SEQ ID NO:1 or fragments thereof. Fragments of SEQ ID NO:1utilized as hybridization probes are preferably greater than 17nucleotides in length and may include nucleotides 358-1684; nucleotides322-459 (encoding LIR conserved sequence); or DNA or RNA sequencescomplementary to SEQ ID NO:5, 6, 23, 24, 27 and 1 or fragments thereof.Fragments of SEQ ID NO:5, 6, 23, 24 and 27 include these sequenceswithout the restrictions sites. Conditions for hybridization may bemoderately stringent conditions described in, for example, Sambrook etal, Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1, pp1.101-104, Cold Spring Harbor Laboratory Press, 1989. Conditions ofmoderate stringency, as defined by Sambrook et al., include use of aprewashing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) andhybridization conditions of about 55° C., 5×SSC, overnight. Highlystringent conditions include higher temperatures of hybridization andwashing. The skilled artisan will recognize that the temperature andwash solution salt concentration may be adjusted as necessary accordingto factors such as the length of the probe. Preferred embodimentsinclude amino acid sequences encoded by DNA that hybridizes to probes ofthe extracellular region of LIR-P3G2 having at least 17 nucleotides.Preferred hybridizing conditions include a temperature of 63° C. for 16hours in a hybridizing solution of Denhart's Solution, 0.05 M TRIS at pH7.5, 0.9 M NaCl, 0.1% sodium pyrophosphate, 1% SDS and 200 μg/mL salmonsperm DNA, followed by washing with 2×SSC at 63° C. for one hour and thea wash with 1×SSC at 63° C. for one hour.

[0037] The present invention includes polypeptides having amino acidsequences that differ from, but are highly homologous to, thosepresented in SEQ ID NO:2, 4, 8, 10, 12, 14, 16, 18, 20 and 22. Examplesinclude, but are not limited to, homologs derived from other mammalianspecies, variants (both naturally occurring variants and those generatedby recombinant DNA technology), and LIR P3G2 and LIR family memberfragments that retain a desired biological activity. Preferably, suchpolypeptides exhibit a biological activity associated with the LIRpolypeptides described in SEQ ID Nos:2, 4, 8, 10, 12, 14, 16, 18 20 and22 and comprise an amino acid sequence that is at least 80% identical toany of the amino acid sequences of the signal peptide and extracellulardomains of the polypeptides presented in SEQ ID NOS:2, 4, 8, 10, 12, 14,16, 18, 20 and 22. Preferably such polypeptides are at least 90%identical to any of the amino acid sequences of the signal peptide andextracellular domains of the polypeptides presented in SEQ ID NOS: 2, 4,8, 10, 12, 14, 16, 18, 20 and 22. Determining the degree of identitybetween polypeptides can be achieved using any algorithms or computerprograms designed for analyzing protein sequences. The commerciallyavailable GAP program described below is one such program Other programsinclude the BESTFIT and GCG programs which are also commerciallyavailable.

[0038] Within the scope of the present invention are LIR polypeptidefragments that retain a desired biological property of an LIRpolypeptide family member such as binding to MHC class I or otherligand. In one such embodiment, LIR polypeptide fragments are solubleLIR polypeptides comprising all or part of the extracellular domain, butlacking the transmembrane region that would cause retention of thepolypeptide on a cell membrane. Soluble LIR polypeptides are capable ofbeing secreted from the cells in which they are expressed.Advantageously, a heterologous signal peptide is fused to the N-terminussuch that the soluble LIR is secreted upon expression. Soluble LIRpolypeptides include extracellular domains incorporating the signalpeptide and those in which the signal peptide is cleaved signal peptide.

[0039] The use of soluble forms of a LIR family member is advantageousfor certain applications. One such advantage is the ease of purifyingsoluble forms from recombinant host cells. Since the soluble proteinsare secreted from the cells, the protein need not be extracted fromcells during the recovery process. Additionally, soluble proteins aregenerally more suitable for intravenous administration and can be usedto block the interaction of cell surface LIR family members with theirligands in order to mediate a desirable immune function.

[0040] Soluble LIR polypeptides include the entire extracellular domainor any desirable fragment thereof, including extracellular domains thatexclude signal peptides. Thus, for example, soluble LIR polypeptidesinclude amino acids x₁-458 of SEQ ID NO:2, where x₁ is amino acids 1 or17; amino acids x₂-459 of SEQ ID NO:4, where x₂ is amino acid 1 or 17;amino acids x₃-439 of SEQ ID NO:8, where x₃ is amino acid 1 or 17; aminoacids x₄-458 of SEQ ID NO:10, where x₄ is amino acid 1 or 17; aminoacids x₅-241 of SEQ ID NO:12, where amino acid x₅ is amino acid 1 or 17,amino acids x₆-461 of SEQ ID NO:14, where x₆ is amino acid 1 or 17;amino acids x₇-449 of SEQ ID NO: 16, where x₇ is amino acid 1 or 17;amino acids x₈-259 of SEQ ID NO:18, where x₈ is amino acid 1 or 17;amino acids x₉-443 of SEQ ID NO:20, where x₉ is amino acid 1 or 17; andamino acids x₁₀-456 of SEQ ID NO:22, where x₁₀ is amino acid 1 or 17.The above identified soluble LIR polypeptides include LIR extracellularregions that include and exclude signal peptides. Additional soluble LIRpolypeptides include fragments of the extracellular domains of familymembers that retain a desired biological activity, such as binding toligands that include MHC class I molecules.

[0041] LIR family member fragments, including soluble polypeptides, maybe prepared by any of a number of conventional techniques. A DNAsequence encoding a desired LIR polypeptide encoding fragment may besubcloned into an expression vector for production of the LIRpolypeptide fragment. The selected encoding DNA sequence advantageouslyis fused to a sequence encoding a suitable leader or signal peptide. Thedesired LIR member encoding DNA fragment may be chemically synthesizedusing known DNA synthesis techniques. DNA fragments also may be producedby restriction endonuclease digestion of a full length cloned DNAsequence, and isolated by electrophoresis on an appropriate gel. Ifnecessary, oligonucleotides that reconstruct the 5′ or 3′ terminus to adesired point may be ligated to a DNA fragment generated by restrictionenzyme digestion. Such oligonucleotides may additionally contain arestriction endonuclease cleavage site upstream of the desired codingsequence, and position an initiation codon (ATG) at the N-terminus ofthe coding sequence.

[0042] Another technique useful for obtaining a DNA sequence encoding adesired protein fragment is the well known polymerase chain reaction(PCR) procedure. Oligonucleotides which define the termini of thedesired DNA are used as probes to synthesize additional DNA from adesired DNA template. The oligonucleotides may also contain recognitionsites for restriction endonucleases, to facilitate inserting theamplified DNA fragment into an expression vector. PCR techniques aredescribed in Saiki et al., Science 239:487(1988): Recombinant DNAMethodology, Wu et al., eds., Academic Press, Inc., San Diego (1989),pp. 189-196; and PCR Protocols: A Guide to Methods and Applications,Innis et al., eds., Academic Press, Inc. (1990).

[0043] DNA of LIR family members of the present invention include cDNA,chemically synthesized DNA, DNA isolated by PCR, genomic DNA, andcombinations thereof. Genomic LIR family DNA may be isolated byhybridization to the LIR family cDNA disclosed herein using standardtechniques. RNA transcribed from LIR family DNA is also encompassed bythe present invention.

[0044] Within the scope of the present invention are DNA fragments suchas LIR polypeptide coding regions and DNA fragments that encode solublepolypeptides. Examples of DNA fragments that encode soluble polypeptidesinclude DNA that encodes entire extracellular regions of LIR familymembers and DNA that encodes extracellular region fragments such asregions lacking the signal peptide. More specifically, the presentinvention includes nucleotides 310-2262 of SEQ ID NO:1 (P3G2 codingregion); nucleotides x₁-1683 of SEQ ID NO:1, where x₁ is 310 or 358(encoding the P3G2 extracellular domain); nucleotides 168-2126 of SEQ IDNO:3 (the 18A3 coding region) and nucleotides x₂-1544 of SEQ ID NO:3,where x₂ is 168 or 216 (the 18A3 extracellular domain coding region);nucleotides x₃-1412 of SEQ ID NO:7, where x₃ is 93 or 141 (the pbm25coding region and extracellular region); nucleotides 184-1980 of SEQ IDNO:9, (the pbm8 coding region) and nucleotides x₄-1557 of SEQ ID NO:9,where x₃ is 184 or 232 (the pmb8 extracellular domain coding region);nucleotides 171-1040 of SEQ ID NO:11 (pbm36-2 coding region) andnucleotides x₅-878 of SEQ ID NO:11, where x₅ is 171 or 219 (encoding thepbm3&2 extracellular domain); nucleotides 183-1652 of SEQ ID NO:13(coding region for pbm36-4) and nucleotides x₆-1565 of SEQ ID NO:13,where x₆ is 183 or 231 (encoding the pbm36-4 extracellular domain);nucleotides 40-1491 of SEQ ID NO:15 (the pbmhh coding region) andnucleotides x₇-1386 of SEQ ID NO:15, where x₇ is 40 or 88 (encoding thepbmhh extracellular domain); nucleotides 30-1376 of SEQ ID NO:17 (thepbm2 coding region) and nucleotides x₈-806 of SEQ ID NO:17, where x₈ is30 or 78 (encoding the pbm2 extracellular region); nucleotides 66-1961of SEQ ID NO:19 (the pbm17 coding region) and nucleotides x₉-1394 of SEQID NO:19, where x₉ is 66 or 114 (encoding the pbm17 extracellulardomain); and nucleotides 67-1839 of SEQ ID NO:21 (the pbmnew codingregion) and nucleotides x₁₀-1434 of SEQ ID NO:21, where x₁₀ is 67 or 115(encoding the pbmnew extracellular domain).

[0045] Included in the present invention are DNAs encoding biologicallyactive fragments of the LIR family members presented in SEQ ID NOS:2, 4,8, 10, 12, 14, 16, 18, 20, and 22.

[0046] The present invention encompasses nucleotide sequences which, dueto the degeneracy of the genetic code, encode polypeptides substantiallyidentical or substantially similar to polypeptides encoded by thenucleic acid sequences described above, and sequences complementary tothem. Accordingly, within the present invention are DNA encodingbiologically active LIR family members which include the coding regionof a native human LIR family member cDNA, or fragments thereof, and DNAwhich is degenerate as a result of the genetic code to the native LIRpolypeptide DNA sequence or the DNA of native LIR family membersdescribed herein.

[0047] In another aspect, the present invention includes LIR variantsand derivatives as well as variants and derivatives of LIR familypolypeptides, both recombinanat and non-recombinant, that retain adesired biological activity. An LIR variant, as referred to herein, is apolypeptide substantially homologous to a native LIR polypeptide, asdescribed herein, except the variant amino acid sequence differs fromthat of the native polypeptide because of one or more deletions,insertions or substitutions.

[0048] LIR family variants may be obtained from mutations of native LIRnucleotide sequences. Within the present invention are such DNAmutations or variants which include nucleotide sequences having one ormore nucleotide additions, nucleotide deletions, or nucleotidesubstitutions compared to native DNA of LIR family members and whichencode variant LIR polypeptides or variant LIR family members having adesired biological activity. Preferably the biological activity issubstantially the same as that of the native LIR polypeptide.

[0049] Variant amino acid sequences and variant nucleotide sequences ofthe present invention preferably are at least 80% identical to that of anative LIR family member sequence. One method for determining the degreeof homology or identity between a native amino acid or nucleotidesequence and a variant amino acid or nucleotide sequence is to comparethe sequences using computer programs available for such purposes. Onesuitable computer program is the GAP program, version 6.0, described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The GAP programutilizes the alignment method of Needleman and Wunsch (J. Mol. Biol.48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482,1981). Briefly, the GAP program defines identity as the number ofaligned symbols (i.e., nucleotides or amino acids) which are identical,divided by the total number of symbols in the shorter of the twosequences being compared. The preferred default parameters for the GAPprogram include: (1) a unary comparison matrix (containing a value of 1for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

[0050] Alterations of native LIR amino acid sequences may be provided byusing any of a number of known techniques. As described above, mutationscan be introduced at selected sequence sites by synthesizingoligonucleotides containing a mutant coding sequence, flanked byrestriction sites enabling its ligation to fragments of the nativesequence. After ligating the synthesized oligonucleotides to the nativesequence fragments, the resulting reconstructed nucleotide sequence willencode an analog or variant polypeptide having the desired amino acidinsertion, substitution, or deletion. Another procedure suitable forpreparing variant polypeptides is oligonucleotide-directed site-specificmutagenesis procedures which provide genes having specific codonsaltered in accordance with the desired substitution, deletion, orinsertion. Techniques for making such alterations include thosedisclosed in the following references: Walder et al. Gene, 42:133, 1986;Bauer et al., Gene 37:73, 1985; Craik, BioTechniques, 12-19 Jan. 1985;Smith et al. Genetic Engineering: Principles and Methods, Plenum Press,1981; and U.S. Pat. Nos. 4,518,584 and 4,737,462, all of which areincorporated herein by reference.

[0051] Variant polypeptides of the present invention may have amino acidsequences which are conservatively substituted, meaning that one or moreamino acid residues of a native LIR polypeptide family member isreplaced by different residues, such that the variant polypeptideretains a desired biological activity that is essentially equivalent tothat of a native LIR family member. In general, a number of approachesto conservative substitutions are well known in the art and can beapplied in preparing variant of the present invention. For example,amino acids of the native polypeptide sequence may be substituted foramino acids which do not alter the secondary and/or tertiary structureof the LIR polypeptide. Other suitable substitutions include those whichinvolve amino acids outside of the ligand-binding domain of interest.One approach to conservative amino acid substitutions involves replacingone or amino acids with those having similar physiochemicalcharacteristics, e.g. substituting one aliphatic residue for anothersuch as Ile, Val, Leu, or Ala for one another); substituting one polarresidue for another (such as between Lys and Arg; Glu and Asp; or Glnand Asn); or substituting entire regions having similar hydrophobicityor hydrophilic characteristics.

[0052] LIR polypeptide variants can be tested for binding to cells asdescribed in Examples 5 and 6 and for phosphatase binding activity asdescribed in Example 11 to confirm biological activity. Other LIRvariants within the present invention include polypeptides which arealtered by changing the nucleotide sequence encoding the polypeptide sothat selected polypeptide Cys residues are deleted or replaced with oneor more alternative amino acids. These LIR variants will not formintramolecular disulfide bridges upon renaturation. Naturally occurringLIR polypeptides selected for alteration by deleting or altering Cysresidues preferably do not have biological activities which depend upondisulfide bridges formed by the Cys residue. Other possible variants areprepared by techniques which cause the modification of adjacent dibasicamino acid residues to enhance expression in yeast systems in which KEX2protease activity is present EP 212,914 discloses site-specificmutagenesis techniques for inactivating KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys and pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

[0053] Naturally occurring LIR variants are also encompassed by thepresent invention. Examples of such variants are proteins that resultfrom alternative mRNA splicing events or from proteolytic cleavage of anLIR polypeptide. Alternative splicing of mRNA may yield a truncated butbiologically active LIR polypeptide such as a naturally occurringsoluble form of the protein. Variations attributable to proteolysisinclude difference in the N- or C-termini upon expression in differenttypes of host cells, due to proteolytic removal of one or more terminalamino acids from the LIR polypeptide. In addition, proteolytic cleavagemay release a soluble form of LIR from a membrane-bound form of thepolypeptide. Other naturally occurring LIR variations are those in whichdifferences from the amino acid sequence of SEQ ID Nos:2, 4, 8, 10, 12,14, 16, 18, 20 and 22 are attributable to genetic polymorphism, theallelic variation among individuals.

[0054] Within the scope of the present invention are derivative LIRfamily polypeptides which include native or variant LIR polypeptidesmodified to form conjugates with selected chemical moieties. Theconjugates can be formed by covalently linking another moiety to anative or variant LIR or by non-covalently linking another moiety to anative or variant LIR. Suitable chemical moieties include but are notlimited to glycosyl groups, lipids, phosphates, acetyl groups, and otherproteins or fragments thereof. Techniques for covalently linkingchemical moieties to proteins are well known in the art and aregenerally suitable for preparing derivative LIR polypeptides. Forexample, active or activated functional groups on amino acid side chainscan be used as reaction sites for covalently linking a chemical moietyto a LIR polypeptide. Similarly, the N-terminus or C-terminus canprovide a reaction site for a chemical moiety. LIR polypeptides orfragments conjugated with other proteins or protein fragments can beprepared in recombinant culture as N-terminal or C-terminal fusionproducts. For example, the conjugate or fusion portions may include asignal or leader sequence attached to an LIR molecule at its N-terminus.The signal or leader peptide co-translationally or post-translationallydirects transfer of the conjugate from its site of synthesis to a siteinside or outside of the cell membrane.

[0055] One useful LIR polypeptide conjugate is one incorporating apoly-His or the antigenic identification peptides described in U.S. Pat.No. 5,011,912 and in Hopp et al., Bio/Technology 6:1124, 1988. Forexample, the FLAG® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK)is highly antigenic and provides an epitope reversibly bound by aspecific monoclonal antibody, thus enabling rapid assay and facilepurification of expressed recombinant protein. This sequence isspecifically cleaved by bovine mucosal enterokinase at the residueimmediately following the Asp-Lys pairing. Fusion proteins capped withthis peptide may be resistant to intracellular degradation in E. coli.Murine hybridoma designated 4E11 produced a monoclonal antibody thatbinds the peptide DYKDDDDK in the presence of certain divalent metalcations, and has been deposited with the American Type CultureCollection under accession no HB 9259. Expression systems useful forproducing recombinant proteins fused to the FLAG® peptide, andmonoclonal antibodies that bind the peptide and are useful in purifyingthe recombinant proteins, are available from Eastman Kodak Company;Scientific Imaging Systems, New Haven, Conn.

[0056] Particularly suitable LIR fusion proteins are those in which anLIR polypeptide is in the form of an oligomer. Oligomers may be formedby disulfide bonds between cysteine residues on more than one LIRpolypeptide, or by noncovalent interactions between LIR polypeptidechains. In another approach, LIR oligomers can be formed by joining LIRpolypeptides or fragment thereof via covalent or noncovalentinteractions between peptide moieties fused to the LIR polypeptide.Suitable peptide moieties include peptide linkers or spacers, orpeptides that have the property of promoting oligomerization. Leucinezippers and certain polypeptides derived from antibodies are among thepeptides that can promote oligomerization of LIR polypeptides attachedthereto.

[0057] Other LIR fusion proteins which promote oligomer formation arefusion proteins having heterologous polypeptides fused to variousportions of antibody-derived polypeptides (including the Fc domain).Procedures for preparing such fusion proteins are described in Ashkenaziet al. PNAS USA 88:10535, 1991; Byrne et al. Nature 344:667, 1990, andHollenbaugh and Aruffo Current Protocols in Immunology, Supplement 4,pages 10.19.1-10.19.11, 1992; all of which are incorporated herein byreference. Example 1 and Example 5 below describe methods for preparingUL18:Fc and P3G2:Fc fusion proteins, respectively, by fusing P3G2 andUL18 to an Fc region polypeptide derived from an antibody. This isaccomplished by inserting into an expression vector a gene fusionencoding the P3G2:Fc fusion protein and expressing the P3G2:Fc fusionprotein. The fusion proteins are allowed to assemble much like antibodymolecules, whereupon interchain disulfide bonds form between the Fcpolypeptides, yielding divalent P3G2 polypeptide. In a similar approach,P3G2 or any LIR polypeptide may be substituted for the variable portionof an antibody heavy or light chain. If fusion proteins are made withheavy and light chains of an antibody, it is possible to form a LIRoligomer with as many as four LIR regions.

[0058] As used herein, a Fc polypeptide includes native and muteinforms, as well as truncated Fc polypeptides containing the hinge regionthat promotes dimerization. One suitable Fc polypeptide is the native Fcregion polypeptide derived from a human IgG1, which is described in PCTapplication WO 93/10151, hereby incorporated herein by reference.Another useful Fc polypeptide is the Fc mutein described in U.S. Pat.No. 5,457,035. The amino acid sequence of the mutein is identical tothat of the native Fc sequence presented in WO 93/10151, except thatamino acid 19 has been changed from Leu to Ala, amino acid 20 has beenchanged from Leu to Glu, and amino acid 22 has been changed from Gly toAla This mutein Fc exhibits reduced affinity for immunoglobulinreceptors.

[0059] Alternatively, oligomeric LIR polypeptide variants may includetwo or more LIR peptides joined through peptide linkers. Examplesinclude those peptide linkers described in U.S. Pat. No. 5,073,627,incorporated herein by reference. Fusion proteins which include multipleLIR polypeptides separated by peptide linkers may be producedconventional recombinant DNA technology.

[0060] Another method for preparing oligomeric LIR polypeptide variantsinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were first identified in several DNA-binding proteins(Landschulz et al. Science 240:1759, 1988). Among the known leucinezippers are naturally occurring peptides and peptide derivatives thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric LIR polypeptides or oligomeric polypeptidesof the LIR family are those described in PCT application WO 94/10308,incorporated herein by reference. Recombinant fusion proteins having asoluble LIR polypeptide fused to a peptide that dimerizes or trimerizesin solution may be expressed in suitable host cells, and the resultingsoluble oligomeric LIR polypeptide recovered from the culturesupernatant.

[0061] Numerous reagents useful for cross-linking one protein moleculeto another are known. Heterobifunctional and homobifunctional linkersare available for this purpose from Pierce Chemical Company, Rockford,Ill., for example. Such linkers contain two functional groups (e.g.,esters and/or maleimides) that will react with certain functional groupson amino acid side chains, thus linking one polypeptide to another.

[0062] One type of peptide linker that may be employed in the presentinvention separates polypeptide domains by a distance sufficient toensure that each domain properly folds into the secondary and tertiarystructures necessary for the desired biological activity. The linkeralso should allow the extracellular portion to assume the proper spatialorientation to form the binding sites for ligands.

[0063] Suitable peptide linkers are known in the art, and may beemployed according to conventional techniques. Among the suitablepeptide linkers are those described in U.S. Pat. Nos. 4,751,180 and4,935,233, which are hereby incorporated by reference. A peptide linkermay be attached to LIR polypeptides by any of the conventionalprocedures used to attach one polypeptide to another. The cross-linkingreagents available from Pierce Chemical Company as described above areamong those that may be employed. Amino acids having side chainsreactive with such reagents may be included in the peptide linker, e.g.,at the termini thereof. Preferably, a fusion proteins formed via apeptide linker are prepared by recombinant DNA technology.

[0064] The fusion proteins of the present invention include constructsin which the C-terminal portion of one protein is fused to the linkerwhich is fused to the N-terminal portion of another protein. Peptideslinked in such a manner produce a single protein which retains thedesired biological activities. The components of the fusion protein arelisted in their order of occurrence (i.e., the N-terminal polypeptide islisted first, followed by the linker and then the C-terminalpolypeptide).

[0065] A DNA sequence encoding a fusion protein is constructed usingrecombinant DNA techniques to insert separate DNA fragments encoding thedesired proteins into an appropriate expression vector. The 3′ end of aDNA fragment encoding one protein is ligated (via the linker) to the 5′end of the DNA fragment encoding another protein with the reading framesof the sequences in phase to permit translation of the mRNA into asingle biologically active fusion protein. A DNA sequence encoding anN-terminal signal. sequence may be retained on the DNA sequence encodingthe N-terminal polypeptide, while stop codons, which would preventread-through to the second (C-terminal) DNA sequence, are eliminated.Conversely, a stop codon required to end translation is retained on thesecond DNA sequence. DNA encoding a signal sequence is preferablyremoved from the DNA sequence encoding the C-terminal polypeptide.

[0066] A DNA sequence encoding a desired polypeptide linker may beinserted between, and in the same reading frame as, the DNA sequencesencoding the two proteins using any suitable conventional technique. Forexample, a chemically synthesized oligonucleotide encoding the linkerand containing appropriate restriction endonuclease cleavage sites maybe ligated between the sequences encoding Fc and a P3G2 polypeptide.

[0067] Within the scope of the present invention are recombinantexpression vectors for expressing polypeptides of the LIR family, andhost cells transformed with the expression vectors. Expression vectorsof the invention include DNA encoding LIR family members operably linkedto suitable transcriptional or translational regulatory nucleotidesequences, such as those derived from a mammalian, microbial, viral, orinsect gene. Examples of regulatory sequences include transcriptionalpromoters, operators, or enhancers, an mRNA ribosomal binding site, andappropriate sequences which control transcription and translationinitiation and termination. Nucleotide sequences are operably linkedwhen the regulatory sequence functionally relates to the LIR DNAsequence. Thus, a promoter nucleotide sequence is operably linked to aLIR DNA sequence if the promoter nucleotide sequence controls thetranscription of the LIR DNA sequence. An origin of replication thatconfers the ability to replicate in the desired host cells, and aselection gene by which transformants are identified, are generallyincorporated in the expression vector.

[0068] In addition, a sequence encoding an appropriate signal peptidecan be incorporated into expression vectors. A DNA sequence for a signalpeptide (secretory leader) may be fused in frame to the LIR sequence sothat the LIR is initially translated as a fusion protein comprising thesignal peptide. A signal peptide that is functional in the intended hostcells promotes extracellular secretion of the LIR polypeptide. Thesignal peptide is cleaved from the LIR polypeptide upon secretion of theLIR polypeptide from the cell.

[0069] Suitable host cells for expression of LIR polypeptides includeprokaryotes, yeast or higher eukaryotic cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. ConingVectors: A Laboratory Manual, Elsevier, N.Y., (1985). Cell-freetranslation systems could also be employed to produce P3G2 polypeptidesusing RNAs derived from DNA constructs disclosed herein.

[0070] Prokaryote host cells suitable in the practice of the presentinvention include gram negative or gram positive organisms, for example,E. coli or Bacilli. Suitable prokaryotic host cells for transformationinclude, for example, E. coli, Bacillus subtilis, Salmonellatyphimurium, and various other species within the general Pseudomonas,Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E.coli, a P3G2 polypeptide may include an N-terminal methionine residue tofacilitate expression of the recombinanat polypeptide. The N-terminalMet may be cleaved from the expressed recombinant LIR polypeptide.

[0071] Expression vectors for use in prokaryotic host cells generallyinclude one or more phenotypic selectable marker genes. A phenotypicselectable marker gene is, for example, a gene encoding a protein thatconfers antibiotic resistance or that supplies an autotrophicrequirement. Examples of useful expression vectors for prokarytoic hostcells include those derived from commercially available plasmids such asthe cloning vector pBR322 (ATCC 37017). pBR322 contains genes forampicillin and tetracycline resistance and thus provides simple meansfor identifying transformed cells. An appropriate promoter and a LIRfamily DNA may be inserted into the pBR322 vector. Other commerciallyavailable vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wis.,USA).

[0072] Promoter sequences commonly used for recombinant prokaryotic hostcell expression vectors include β-lactamase (penicillinase), lactosepromoter system (Chang et al. Nature 75:615, 1978; and Goeddel et al.,Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al.,Nucl. Acids Res. 8:4057, 1980); and EP-A-36776) and tac promoter(Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, p. 412, 1982). A particularly useful prokaryotic host cellexpression system employs a phase λP_(L) promoter and a cI857tsthermolabile repressor sequence. Plasmid vectors available from theAmerican Type Culture Collection which incorporate derivatives of theλP_(L) promoter include plastid pHUB2 (resident in E. coli strain JMB9,ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).

[0073] Alternatively, LIR polypeptides may be expressed in yeast hostcells, preferably from the Saccharomyces genus (e.g., S. cerevisiae).Other genera of yeast, such as Pichia or Kluyveromyces may also beemployed. Yeast vectors will often contain an origin of replicationsequence from a 2μ yeast plasmid, an autonomously replicating sequence(ARS), a promoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968); and Holland et al., Biochem. 17:4900,1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,675. Anotheralternative is the glucose-repressible ADH2 promoter described byRussell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature300:724, 1982). Shuttle vectors replicable in both yeast and E. coli maybe constructed by inserting DNA from pBR322 for selection andreplication in E. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

[0074] The yeast α-factor leader sequence may be employed to directsecretion of the LIR polypeptide. The α-factor leader sequence is ofteninserted between the promoter sequence and the structural gene sequence.See, e.g., Kurjan et al., Cell 30:933,1982 and Bitter et al., Proc.Natl. Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence may be modifiednear its 3′ end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

[0075] Yeast transformation protocols are known to those of skill in theart. One such protocol is described by Hinnen et al., Proc. Natl. Acad.Sci. USA 75: 1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 μg/mL adenine and 20 μg/mL uracil.

[0076] Yeast host cells transformed by vectors containing an ADH2promoter sequence may be grown for inducing expression in a “rich”medium. An example of a rich medium is one having 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/mL uracil. Derepressionof the ADH2 promoter occurs when glucose is exhausted from the medium.

[0077] Mammalian or insect host cell culture systems may be used toexpress recombinant LIR polypeptides. Baculovirus systems for productionof heterologous proteins in insect cells are reviewed by Luckow andSummers, Bio/Technology 6:47 (1988). Established cell lines of mammalianorigin also may be employed. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL1651)(Gluzman et al, Cell 23:175, 1981), L cells, C127 cells, 3T3 cells(ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK(ATCC CRL 10) cell lines, and the CVI/EBNA cell cline derived from theAfrican green monkey cell line CVI (ATCC CCL 70) as described by McMahanet al. (EMBO J. 10:2821, 1991). COS-1 (ATCC CRL-1650).

[0078] Transcriptional and translational control sequences for mammalianhost cell expression vectors may be excised from viral genomes. Commonlyused promoter sequences and enhancer sequences are derived from Polyomavirus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.DNA derived from the SV40 viral genome, for example, SV40 origin, earlyand late promoter, enhancer, splice, and polyadenylation sites may beused to provide other genetic elements for expression of a structuralgene sequence in a mammalian host cell. Viral early and late promotersare particularly useful because both are easily obtained from a viralgenome as a fragment which may also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978). Smaller or larger SV40fragments may also be used, provided the approximately 250 bp sequenceextending from the HIND III site toward the Bg/I site located in theSV40 viral origin of replication site is included.

[0079] Suitable expression vectors for use in mammalian host cells canbe constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280,1983). One useful system for stable high level expression of mammalianreceptor cDNAs in C127 murine mammary epithelial cells can beconstructed substantially as described by Cosman et al. (Mol. Immunol.23:935, 1986). A high expression vector, PMLSV N1/N4, described byCosman et al., Nature 312:768, 1984 has been deposited as ATCC 39890.Additional mammalian expression vectors are described in EP-A-0367566,and in WO 91/18982. Still additional expression vectors for use inmammalian host cells include pDC201 (Sims et al., Science 241:585,1988), pDC302 (Mosley et al. Cell 59:335, 1989), and pDC406 (McMahan etal., EMBO J. 10:2821, 1991). Vectors derived from retroviruses also maybe employed. One preferred expression system employs pDC409 as discussedin Example 5 below.

[0080] For expression of LIR polypeptides the expression vector maycomprise DNA encoding a signal or leader peptide. In place of the nativesignal sequence, a heterologous signal sequence may be added, such asthe signal sequence for interleukin-7 (IL-7) described in U.S. Pat. No.4,965,195; the signal sequence for interleukin-2 receptor described inCosman et al., Nature 312:768, 1984); the interleukin-4 signal peptidedescribed in EP 367,566; the type I interleukin-1 receptor signalpeptide described in U.S. Pat. No. 4,968,607; and the type IIinterleukin-1 receptor signal peptide described in EP 460,846.

[0081] Further contemplated within the present invention are purifiedLIR family polypeptides. The purified polypeptides of the presentinvention may be purified from recombinant expression systems asdescribed above or purified from naturally occurring cells. The desireddegree of purity may depend on the intended use of the protein with arelatively high degree of purity preferred when the protein is intendedfor in vivo use. Preferably, LIR polypeptide purification processes aresuch that no protein bands corresponding to proteins other than thedesired LIR protein are detectable by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). It will be recognized by one skilled in theart that multiple bands corresponding to any LIR polypeptide my bedetected by SDS-PAGE, due to differential glycosylation, variations inpost-translational processing, and the like, as discussed above. Mostpreferably, any specific LIR polypeptide is purified to substantialhomogeneity, as indicated by a single protein band upon analysis bySDS-PAGE. The protein band may be visualized by silver staining,Coomassie blue staining, or by autoradiography or fluorescence if theprotein is appropriately labeled.

[0082] One process for providing purified LIR polypeptides includesfirst culturing a host cell transformed with an expression vectorcomprising a DNA sequence that encodes the desired polypeptide underconditions that promote expressing the desired LIR polypeptide and thenrecovering the LIR polypeptide. As the skilled artisan will recognize,procedures for recovering the polypeptide will vary according to suchfactors as the type of host cells employed and whether the polypeptideis secreted in the culture medium is extracted from cells.

[0083] When the expression system secretes the polypeptide into theculture medium, the medium may be first concentrated using acommercially available protein concentration filter, for example, anAmicon or Millipore Pellicon ultrafiltration unit. Following theconcentration step, the concentrate can be applied to a suitablepurification matrix such as a gel filtration medium. Alternatively, ananion exchange resin can be employed, such as a resin matrix or resinsubstrate having pendant diethylaminoethyl (DEAE) groups. The matricescan be acrylamide, agarose, dextran, cellulose or other types commonlyemployed in protein purification. Similarly, a purification matrixhaving cation exchange groups such as sulfopropyl or carboxymethylfunctionalities on an insoluble matrix can be used. Sulfopropyl groupsare preferred. Still other purification matrices and methods suitablefor providing purified LIR are high performance liquid chromatographyusing hydrophobic reversed phase media (RP-HPLC). One skilled in the artwill recognized the any or all of the foregoing purification steps, invarious combinations, can be employed to provide a purified LIRpolypeptide.

[0084] Alternatively, LIR polypeptides can be purified by immunoaffinitychromatography. An affinity column containing an antibody that binds aLIR polypeptide may be prepared by conventional procedures and employedin purifying LIR. Example 5 describes a procedures for generatingmonoclonal antibodies directed against P3G2 which may be utilized inimmunoaffinity chromatography.

[0085] Recombinant protein produced in bacterial culture may be isolatedby first disrupting the host cells by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents and then extracting the polypeptide from cell pellets ifthe polypeptide is insoluble, or from the supernatant fluid if thepolypeptide is soluble. After the initial isolation step, thepurification process may include one or more concentrating, salting out,ion exchange, affinity, or size exclusion chromatography purificationsteps. For many application a final RP-HPLC purification step isbeneficial.

[0086] Additional methods for providing LIR polypeptides and purifiedLIR polypeptides involves fermenting yeast which express proteins as asecreted protein. Secreted recombinant protein resulting from alarge-scale fermentation can be purified by methods analogous to thosedisclosed by Urdal et al. (J. Chromatog. 296:171, 1984), involving twosequential, reversed-phase HPLC steps for purification of a recombinantprotein on a preparative HPLC column.

[0087] LIR-P3G2 DNA in pDC406 vector was deposited with the AmericanType Culture Collection on Apr. 22, 1997 and assigned accessionNo.______. The deposit was made under the terms of the Budapest Treaty.

[0088] As described above, LIR-P3G2 is a MHC class I receptor moleculefound on the surface of certain monocytes, B cells, and NK cells.Certain LIR family members have the ITIM motif and by analogy with thestructure and function of known MHC class I receptor molecules, areinhibitory receptors mediating negative signaling. Other LIR familymembers lack the ITIM motif and by analogy with the structure andfunction of known MHC class I receptors are activatory receptors.Failure of a receptor that mediates negative signaling could result inautoimmune diseases. Thus, engaging an LIR family member having ITIMmotifs with an agonistic antibody or ligand can be used to downregulatea cell function in disease states in which the immune system isoveractive and excessive inflammation or immunopathology is present. Onthe other hand, using an antagonistic antibody specific to the ITIMpossessing LIR receptor or a soluble form of the receptor can be used toblock the interaction of the cell surface receptor with the receptor'sligand to activate the specific immune function in disease statesassociated with suppressed immune function. Since receptors lacking theITIM motif send activatory signals once engaged as described above,failure of a receptor that mediates an activatory signal could result insuppressed immune function. Engaging the receptor with its agonisticantibody or ligand can be used to treat diseases associated with thesuppressed immune function. Using an antagonistic antibody specific tothe activatory LIR receptor or a soluble form of the receptor can beused to block the interaction of the activatory receptor with thereceptor's ligand to downregulate the activatory signaling.

[0089] Since LIR-P3G2 binds to various cells, LIR-P3G2 may be used topurify or isolate these cells from heterogeneous preparations.Additionally, P3G2 probes can be used to isolate and identify relatedmolecules.

[0090] LIR polypeptides of the present invention may be used indeveloping treatments for any disorder mediated directly or indirectlyby defective or insufficient amounts of any of the LIR polypeptides. Atherapeutically effective amount of purified LIR protein is administeredby a patient afflicted with such a disorder. Alternatively, LIR DNA maybe employed in developing a gene therapy approach to treating suchdisorders. Disclosure herein of native LIR nucleotide sequence permitsthe detection of defective LIR genes, and the replacement thereof withnormal LIR-encoding genes. Defective genes may be detected in vitrodiagnostic assays, and by comparison of the native LIR nucleotidesequence disclosed herein with that of an LIR gene derived from a personsuspected of harboring a defect in the gene.

[0091] The present invention also provides pharmaceutical compositionswhich may include an LIR polypeptide, or fragments or variants thereofwith a physiologically acceptable carrier or diluent. Such carriers anddiluents will be nontoxic to recipients at the dosages andconcentrations employed. Such compositions may further include buffers,antioxidants such as ascorbic acid, low molecular weight (less thanabout ten residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients commonly used inpharmaceutical compositions. The pharmaceutical compositions of thepresent invention may be formulated as a lyophilizate using appropriateexcipient solutions as diluents. The pharmaceutical compositions mayinclude an LIR polypeptide in any for described herein, including butnot limited to active variants, fragments, and oligomers. LIRpolypeptides may be formulated according to known methods that are usedto prepare pharmaceutically useful compositions. Components that arecommonly employed in pharmaceutical formulations include those describedin Remington's Pharmaceutical Sciences, 16th ed. (Mack PublishingCompany, Easton, Pa., 1980).

[0092] The pharmaceutical preparations of the present invention may beadministered to a patient, preferably a human, in a manner appropriateto the indication. Thus, for example, the compositions can beadministered by intravenous injection, local administration, continuousinfusion, sustained release from implants, etc. Appropriate dosages andthe frequency of administration will depend on such factors as thenature and severity of the indication being treated, the desiredresponse, the condition of the patient and so forth.

[0093] In preferred embodiments an LIR polypeptide used in thepharmaceutical compositions of the present invention is purified suchthat the LIR polypeptide is substantially free of other proteins ofnatural or endogenous origin, desirably containing less than about 1% bymass of protein contaminants residual of the production processes. Suchcompositions, however, can contain other proteins added as stabilizers,carriers, excipients or co-therapeutics.

[0094] LIR encoding DNAs and DNA fragments disclosed herein find use inthe production of LIR polypeptides, as described above. In oneembodiment, such fragments comprise at least about 17 consecutivenucleotides, more preferably at least 30 consecutive nucleotides, of LIRDNA. DNA and RNA complements of the fragments have similar utility.Among the uses of LIR nucleic acid fragments are as probes or primers inpolymerase chain reactions. For example, a probe corresponding to afragment of DNA encoding the extracellular domain of LIR may be employedto detect the presence of LIR nucleic acids in in vitro assays and inother probing assays such as Northern Blot and Southern blot assays.Cell types expressing an LIR polypeptide can be identified using LIRfamily nucleic acid probes using probing procedures well known in theart. Those skilled in the art have the knowledge to choose a probe ofsuitable length and apply conventional PCR techniques to isolate andamplify a DNA sequence.

[0095] Nucleic acid fragments may also be used as a probe in crossspecies hybridization procedures to isolate LIR, DNA from othermammalian species. As one example, a probe corresponding to theextracellular domain of an LIR polypeptide may be employed. The probesmay be labeled (e.g., with ³²P) by conventional techniques.

[0096] Other useful fragments of LIR nucleic acids are antisense orsense oligonucleotides which include a single-stranded nucleic acidsequence (either RNA or DNA) capable of binding to a target LIR mRNA(sense) or P3G2 DNA (antisense) sequences. Such fragments are generallyat least about 14 nucleotides, preferably from about 14 to about 30nucleotides. The ability to create an antisense or a senseoligonucleotide based upon a cDNA sequence for a given protein isdescribed in, for example, Stein and Cohen, Cancer Res. 48:2659, 1988and van der Krol et al., BioTechniques 6:958, 1988.

[0097] Binding antisense or sense oligonucleotides to target nucleicacid sequences results in the formation of duplexes that blocktranslation (RNA) or transcription (DNA) by one of several means,including enhanced degradation of the duplexes, premature termination oftranscription or translation, or by other means. The antisenseoligonucleotides thus may be used to block LIR expression.

[0098] In one embodiment antisense or sense LIR oligonucleotides used inbinding procedures may encompass oligonucleotides having modifiedsugar-phosphodiester backbones (or other sugar linkages, such as thosedescribed in WO91/06629) and wherein such sugar linkages are resistantto endogenous nucleases. Oligonucleotides having sugar linkagesresistant to endogenous nucleases are stable in vivo (i.e., capable ofresisting enzymatic degradation) but retain sequence specificity to beable to bind to target nucleotide sequences. Other examples of sense orantisense oligonucleotides include those oligonucleotides which arecovalently linked to organic moieties, such as those described in WO90/10448, and other moieties that increase affinity of theoligonucleotide for a target nucleic acid sequence, such aspoly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

[0099] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO₄-mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. Antisense or sense oligonucleotides are preferably introducedinto a cell containing the target nucleic acid sequence by inserting heantisense or sense oligonucleotide into a suitable retroviral vector,then contacting the cell with the retroviral vector containing theinserted sequence, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see PCT Application U.S.90/02656).

[0100] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors. Preferably, conjugating the ligandbinding molecule does not substantially interfere with the ability ofthe ligand binding molecule to bind its corresponding molecule orreceptor, or block entry of the sense of antisense oligonucleotide orits conjugated version into the cell.

[0101] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0102] In still a further aspect, the present invention providesantibodies that specifically bind LIR polypeptides, i.e., antibodiesbind to LIR polypeptides via an antigen-binding site of the antibody (asopposed to non-specific binding). Antibodies of the present inventionmay be generated using LIR polypeptides or immunogenic fragmentsthereof. Polyclonal and monoclonal antibodies may be prepared byconventional techniques. See, for example, Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al.(eds.), Plenum Press, New York 1980; and Antibodies: A LaboratoryManual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988. An exemplary procedure for producingmonoclonal antibodies immunoreactive with P3G2-LIR is furtherillustrated in Example 5 below.

[0103] Included within the scope of the present invention are antigenbinding fragments of antibodies which specifically bind to an LIRpolypeptide. Such fragments include, but are not limited to, Fab,F(ab′), and F(ab′)₂. Antibody variants and derivatives produced bygenetic engineering techniques are contemplated as within the presentedinvention.

[0104] The monoclonal antibodies of the present invention includechimeric antibodies, e.g., humanized versions of murine monoclonalantibodies. Such antibodies may be prepared by known techniques andoffer the advantage of reduced immunogenicity when the antibodies areadministered to humans. In one embodiment a humanized monoclonalantibody comprises the variable region of a murine antibody (or just theantigen binding site thereof) and a constant region derived from a humanantibody. Alternatively, a humanized antibody fragment may comprise theantigen binding site of a murine monoclonal antibody and a variableregion fragment (lacking the antigen-binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal., Nature 332:232, 1988; Lie et al. PNAS 84:3439, 1987; Larrick et al.Bio/Technology 7:934, 1989; and Winter and Harris TIPS 14:139, 1993.

[0105] As mentioned above, antibodies of the present invention areuseful in in vitro or in vivo assays to detect the presence of LIRpolypeptides and in purifying an LIR polypeptide by affinitychromatography.

[0106] Additionally, antibodies capable of blocking an LIR from bindingto target cells may be used to inhibit a biological activity of an LIRpolypeptide. More specifically, therapeutic compositions of an antibodyantagonistic to one or more LIR family members having the ITIM motif maybe administered to an individual in order to block the interaction of acell surface LIR with its ligand. The result is an activation of immunefunction and is particularly beneficial in disease states in which theimmune system is hyporesponsive or suppressed. Conversely, therapeuticcompositions of an antibody antagonistic to one or more LIR familymembers lacking the ITIM motif may be used to obtain the opposite effectand be beneficial in disease states in which the immune system isoveractive and excessive inflammation or immunopathology is present.

[0107] Pharmaceutical compositions which include at least one antibodythat is immunoreactive with an LIR polypeptide and a suitable diluent,excipient, or carrier, are considered with the present invention.Suitable diluents, excipients, and carriers are described in the contextof pharmaceutical compositions which include polypeptides of the presentinvention.

[0108] The following examples are provided to illustrate certainembodiments of the invention, and are not to be construed as limitingthe scope of the invention.

EXAMPLES Example 1 Isolating and Expressing Viral Protein

[0109] DNA encoding P3G2 polypeptide of the present invention wasidentified by isolating and expressing a viral glycoprotein, UL18, knownto be expressed on cells infected with HCMV, and then expressing andusing a UL18/Fc fusion protein to search for UL18 receptors. DNAencoding UL18 and its amino acid sequence are known and described inBeck, S., B. G. Barrell, Nature 331:269-272, 1988. The followingdescribes isolating UL18 and preparing the UL18/Fc fusion protein.

[0110] Using standard techniques, total RNA was isolated from HumanForeskin Fibroblasts infected with HCMV (AD169) at three differenttranscription stages-immediate early (IE, 8 p.i.h.), early (24 p.i.h.)and late (48 p.i.h.). Because UL18 is known to be transcribed early inthe infection, the IE total RNA was polyA+ selected and used toconstruct an HCMV-IE cDNA Library using a cDNA kit according to themanufacturer's instructions (Pharmacia TIME SAVER cDNA Kit). In order toisolate the full length UL18 gene, two oligonucleotide primers known toinclude the terminal sequences of the UL18 gene were synthesized andused to isolate and amplify the UL18 gene from the HCMV-IE cDNA library.The primers had the following sequences and included Not I restrictionsites which incorporate into the PCR product.            Not I 5′ - TATGCG GCC GCC ATG ATG ACA ATG TGG T - 3′ (SEQ ID NO:23) 5′ - TATGCG GCC GCC CCT TGC GAT AGC G - 3′ (SEQ ID NO:24)            Not I

[0111] The PCR conditions included one 5 minute 95° C. cycle followed by30 cycles of 45 seconds at 95°, 45 seconds at 58° and 45 seconds at 72°,and then one cycle for 5 minutes at 72° C. The PCR product waselectrophoresed on a 1% agarose gel and sized using ethidium bromide tovisualize the separated DNA products. The presence of DNA of having theexpected size of approximately 1.1 kb was confirmed.

[0112] The pDC409 expression vector, a vector derived from pDC406(McMahan et al., EMBO J. 10:2821, 1991) but having a single Bgl II sitewas selected for the cloning process. The PCR product was subcloned intoa pDC409 expression vector through the Not I sites, sequenced and theamino acid sequence deduced from the DNA sequence. The determinednucleotide sequence and amino acid sequence were identical to thepreviously published sequences (ibid.).

[0113] A fusion protein of the extracellular region of UL18 and a muteinhuman IgG1 Fc region (UL18:Fc) was prepared by first isolating cDNAencoding the extracellular region of UL18 using primers which flank theextracellular region of UL18. The primers were synthesized with Sal Iand Bgl II restriction sites inserted at the 5′ and 3′ termini so thatthe PCR amplified cDNA introduced Sal I and Bgl II restriction sites atthe 5′ and 3′ ends, respectively. The primers had the followingsequences: 5′ - ATA GTC GAC AAC GCC ATG ATG ACA ATG TGG TG - 3′ (SEQ IDNO:25)          Sal I 5′ - TAA AGA TCT GGG CTC GTT AGC TGT CGG GT - 3′(SEQ ID NO:26)           Bgl II

[0114] The conditions for the PCR reaction were as described aboveexcept that the template was the full length gene isolated as justdescribed.

[0115] To prepare a vector construct for expressing fusion protein,sUL18:Fc, for use in cell binding studies, a DNA fragment encoding theFc region of a human IgG1 antibody was isolated from a plasmid using BglII and Not I restriction enzymes. The encoded Fc portion was the muteinFc described in U.S. Pat. No. 5,457,035 having reduced affinity forimmunoglobulin receptors. The Bgl II site on the sUL18 gene was used toligate the sUL18 gene DNA to the Bgl II site on the Fc gene to form asUL18:Fc fusion DNA construction having an N-terminal Sal I restrictionsite and a C-terminal Not I restriction site. This fusion sUL18:Fc DNAconstruct was then ligated into pDC409 expression vector at its Sal Iand Not I sites to form a 409/sUL18/Fc DNA construct.

[0116] The monkey cell line COS-1 (ATCC CRL-1650) was used to confirmexpression of the fusion protein. COS-1 cells in 6-well plates (2×10⁵cells per well) were transfected with about 2 μg of the DNA construct409/sUL18/Fc per well. The cells were cultured for 2-3 days in 5%FBSDMEM/F12 (available from GIBCO), then washed twice with PBS, starvedfor 1 hour in cysteine/methionine depleted RPMI (available from GIBCO asRPMI 1640) and metabolically labeled with 100 μCi/mL of ³⁵S-Met/Cys for4 hours. The supernatant was spun clear to remove loose cells and 150 μLof the supernatant was incubated with 100 μL of RIPA (0.05% Tween 20,0.1% SDS, 1% Triton X-100, 0.5% deoxycholate in PBS) buffer and 50 μL of50% Protein A-Sepharose solid support beads at 4° C. for 1 hour. ProteinA-Sepharose is a Sepharose solid support (available from Pharmacia)having immobilized Protein A which binds the Fc portion of the fusionprotein. After washing the solid support with RIPA to remove unboundmaterial, fusion protein bound to the Protein A-Sepharose solid supportwas eluted from the Protein A-Sepharose using 35 μL of SDS-PAGE reducingsample buffer and then heated at 100° C. for 5 minutes. The eluant wasthen electrophoresed on a 4-20% SDS polyacrylamide gradient gel with ¹⁴Clabeled protein molecular weight markers. After electrophoresis the gelwas fixed with 8% acetic acid and enhanced at room temperature for 20minutes with Amplifier available from Amersham. After drying the gelunder vacuum it was exposed to x-ray film. Film analysis confirmed thatthe expected protein, a 100-120 kDa protein which includes the mutein Fcregion of IgG and UL18 extracellular domains fused to the Fc, wasexpressed.

[0117] Once cells expressing the fusion protein were identified largescale cultures of transfected cells were grown to accumulate supernatantfrom cells expressing the fusion protein. This procedure involvedtransfecting COS-1 cells in T175 flasks with 15 μg of the UL18/Fc/409fusion DNA per flask. After 7 days of culture in medium containing 0.5%low immunoglobulin bovine serum, a solution of 0.2% azide was added tothe supernatant and the supernatant was filtered through a 0.22 μmfilter. Then approximately 1 L of culture supernatant was passed througha BioCad Protein A HPLC protein purification system using a 4.6×100 mmProtein A column (POROS 20A from PerSeptive Biosystems) at 10 mL/min.The Protein A column binds the Fc portion of the sUL18/Fc fusion proteinin the supernatant, immobilizing the fusion protein and allowing othercomponents of the supernatant to pass through the column. The column waswashed with 30 mL of PBS solution and bound sUL18/Fc was eluted from theHPLC column with citric acid adjusted to pH 3.0. Eluted purifiedsUL18/Fc was neutralized as it eluted using 1M Hepes solution at pH 7.4.The pooled eluted protein was analyzed using SDS PAGE with silverstaining, confirming expression of the 100-120 kDa UL18/Fc fusionprotein.

Example 2 Screening Cell Lines for Binding to UL18

[0118] The sUL18/Fc protein isolated as described in Example 1 was usedto screen cells lines to which it binds using quantitative bindingstudies according to standard flow cytometry methodologies. For eachcell line screened, the procedure involved incubating approximately100,000 of the cells blocked with 2% FCS (fetal calf serum), 5% normalgoat serum and 5% rabbit serum in PBS for 1 hour. Then the blocked cellswere incubated with 5 μg/mL of sUL18/Fc fusion protein in 2% FCS, 5%goat serum and 5% rabbit serum in PBS. Following the incubation thesample was washed 2 times with FACS buffer (2% FCS in PBS) and thentreated with mouse anti human Fc/biotin (purchased from JacksonResearch) and SAPE (streptavidin-phycoerythrin purchased from MolecularProbes). This treatment causes the anti human Fc/biotin to bind to anybound sUL18/Fc and the SAPE to bind to the anti human Fc/biotinresulting in a fluorescent identifying label on sUL18/Fc which is boundto cells. The cells were analyzed for any bound protein usingfluorescent detection flow cytometry. The results indicated that UL18binds well to B cell lines CB23, RAJI and MP-1; monocytic cell linesThp-1 and U937; and primary B cell and primary monocytes. UL18 does notbind detectably to T cell lines nor does it bind to primary T cells.

Example 3 Isolating a P3G2 cDNA and Polypeptide

[0119] The following describes screening cDNA of one of the cell linesfound to bind UL18 and the isolation of a novel polypeptide expressed bythe cell line. A CB23 cDNA library in the mammalian expression vectorpDC406, prepared as described in U.S. Pat. No. 5,350,683 (incorporatedherein by reference) was obtained and plasmid DNA was isolated frompools consisting of approximately 2,000 clones per pool. The isolatedDNA was transfected into CV1-EBNA cells (ATCC CRL 10478) usingDEAE-dextran followed by chloroquine treatment. The CV1-EBNA cells weremaintained in complete medium (Dulbecco's modified Eagles' mediacontaining 10% (v/v) fetal calf serum, 50 U/mL penicillin, 50 U/mLstreptomycin, and 2 mM L-glutamine) and were plated to a density ofapproximately 2×10⁵ cells/well in single-well chambered slides. Theslides had been pre-treated with 1 mL of a solution of 10 μg/mL humanfibronectin in PBS for 30 minutes followed by a single washing with PBS.Media was removed from adherent cells growing in a layer and replacedwith 1.5 mL complete medium containing 66.6 μM chloroquine sulfate.About 0.2 mL of a DNA solution (2 μg DNA, 0.5 mg/mL DEAE-dextran incomplete medium containing chloroquine) was added to the cells and themixture was incubated at 37 C for about five hours. Followingincubation, the media was removed and the cells were shocked by additionof complete medium containing 10% DMSO (dimethylsulfoxide) for 2.5minutes. Shocking was followed by replacing the solution with freshcomplete medium. The cells were grown in culture for two to three daysto permit transient expression of the inserted DNA sequences. Theseconditions led to a 30% to 80% transfection frequency in survivingCV1-EBNA cells.

[0120] Each slide was incubated with 1 mL of UL18:Fc at a concentrationof 1 μg/mL in binding buffer (RPMI 1640 containing 25 mg/mL bovine serumalbumin, 2 mg/mL sodium azide, 20 mM Hepes at pH 7.2, and 50 mg/mLnonfat dry milk) at room temperature for 1 hour. The incubated slideswere washed with the binding buffer and then incubated with Fc specific¹²⁵I-mouse anti-human IgG (see Goodwin et al., Cell 73:447-456, 1993).This was followed by a second wash with buffer after which the slideswere fixed with a 2.5% glutaraldehyde/PBS solution, washed with PBSsolution and allowed to air dry. The dried slides were dipped in KodakGTNB-2 photographic emulsion (6× dilution in water). After air drying,the slides were placed in a dark box and refrigerated. After three daysthe slides were developed in Kodak D19 developer, rinsed in water andfixed in Agfa G433C fixer. The fixed slides were individually examinedunder a micro cope at 25-40× magnification. Positive cells demonstratingbinding of sUL18:Fc were visualized by the presence of autoradiographicsilver grains against the film background. Two positive pools wereidentified. Bacterial clones from each pool were titered and plated toprovide plates containing approximately 200 colonies each. Each platewas scraped to provide pooled plasmid DNA for transfection into CV1-EBNAcells and screening as described above. Following subsequent breakdownsand screenings, two positive individual colonies were obtained. The cDNAinserts of the two positive clones were 2922 and 2777 nucleotides inlength as determined by automated DNA sequences. The coding regions ofthe two inserts, designated P3G2 and 18A3 were 1953 (nucleotides310-2262) and 1959 (nucleotides 168-2126) nucleotides, respectively. Thetwo cDNA clones encode proteins that are substantially similar andprobably represent different alleles of the same gene.

[0121] The cDNA sequence and encoded amino acid of P3G2 are presented inSEQ ID NO:1 and SEQ ID NO:2, respectively. The cDNA sequence and encodedamino acid of 18A3 are presented in SEQ ID NO:3 and SEQ ID NO:4,respectively. The P3G2 amino acid sequence (SEQ ID NO:2) has a predictedsignal peptide of 16 amino acids (amino acids 1-16); an extracellulardomain of 442 amino acids (amino acids 17-458); a transmembrane domainof 25 amino acids (amino acids 459-483) and, a cytoplasmic domain of 167amino acids (amino acids 484-650. The extracellular domain includes fourimmunoglobulin-like domains. Ig-like domain I includes approximatelyamino acids 17-118; Ig-like domain II includes approximately amino acids119-220; Ig-like domain III includes approximately amino acids 221-318;and Ig-like domain IV includes approximately amino acids 319-419.Significantly, the cytoplasmic domain of this polypeptide includes fourITIM motifs, each having the consensus sequence of YxxL/V. The firstITIM motif pair is found at amino acids 533-536 and 562-565 and thesecond pair is found at amino acids 614-617 and 644-647. The amino acidsequence of 18A3 is nearly identical having the features describesabove.

[0122] The features of these encoded polypeptides are consistent with atype I transmembrane glycoprotein.

Example 4 Preparing P3G2 Fusion Protein

[0123] The following describes procedures used to generate a P3G2 fusionprotein which was then used to identify cell lines to which it binds andfinally isolate a normal cell-surface P3G2 ligand which is distinct fromUL18. A fusion protein of the extracellular region of P3G2 and themutein human Fc region (sP3G2:Fc) was prepared by first isolating cDNAencoding the extracellular region of P3G2 using primers which flank theextracellular region of P3G2. The primers were synthesized with Sal Iand Bgl II restriction sites inserted at the 5′ and 3′ termini so thatthe PCR amplified cDNA introduced Sal I and Bgl II restriction sites atthe 5′ and 3′ ends, respectively. The primers had the followingsequences:            Sal I 5′ - TAT GTC GAC CAT GAC CCC CAT CCT CACGGT - 3′ (SEQ ID NO:5)                                  Bgl II      Xa5′ - TAT GGG CTC TGC TCC AGG AGA AGA TCT TCC TTC TAT AAC (SEQ ID NO:6)   CCC CAG GTG CCT T

[0124] The conditions for the PCR reaction were as described above andthe template was the full length gene P3G2 gene isolated as described inExample 3 above.

[0125] To prepare a vector construct for expressing fusion proteinsP3G2:Fc for use in cell binding studies, the mutein human Fc region ofIgG1 was cut from the plasmid described above in Example 1 using Bgl IIand Not I restriction enzymes. The Bgl II site on the sP3G2 gene wasused to ligate the sP3G2 gene DNA to the Bgl II site on the human muteinFc gene to form a sP3G2/Fc fusion DNA construction having an N-terminalSal I restriction site and a terminal Not I restriction site. Thisfusion sP3G2:Fc DNA construct was then ligated into pDC409 expressionvector at its Sal I and Not I sites to form a 409/sP3G2/Fc DNAconstruct.

[0126] The monkey cell line COS-1 (ATCC CTL-1650) was used to confirmexpression of the fusion protein. COS-1 cells in 6-well plates (2×10⁵cells per well) were transfected with about 2 μg of the DNA construct409/sP3G2/Fc per well. The cells were cultured in 5% FBS/DMEM/F12(available from GIBCO) and at day two or three following transfection,the cells were starved for 1 hour in cysteine/methionine depleted RPMIand the transfected cells were metabolically labeled with 100 μCi/mL of³⁵S-Met/Cys for 4 hours. The supernatant was spun clear to removed loosecells and debris and 150 μL of the supernatant was incubated with 100 μLof RIPA buffer and 50 μL of 50% Protein A-Sepharose solid support beadsat 4° C. for 1 hour. After washing the solid support with RIPA to removeunbound material, fusion protein bound to the Protein A-Sepharose solidsupport was eluted from the Protein A-Sepharose using 30 μL of SDS-PAGEreducing sample buffer and then heated at 100° C. for 5 minutes. Theeluant was then electrophoresed on a 4-20% SDS polyacrylamide gradientgel with ¹⁴C labeled protein molecular weight markers. Afterelectrophoresis the gel was fixed with 8% acetic acid and enhanced atroom temperature for 20 minutes with Amplifier available from Amersham.After drying the gel under vacuum it was exposed to x-ray film. Filmanalysis confirmed that the expected protein, having a molecular weightof 120-130 kDa, was expressed.

[0127] Once fusion protein expression was verified, large scale culturesof transfected cells were grown to accumulate supernatant from COS-1cells expressing the fusion protein as described in Example 1 above. TheP3G2/Fc fusion protein was purified according to the procedure describedin Example 3 above using the BioCad system and the POROS 20A column fromPerSeptive Biosystems. The pooled eluted protein was analyzed using SDSPAGE with silver staining, confirming expression.

Example 5 (Generating LTR-P3G2 Antibody

[0128] The following example describes generating monoclonal antibody toP3G2 that was used in flow cytometry analysis to identify cells on whichP3G2 is expressed. Purified P3G2/Fc fusion protein was prepared by COS-1cell expression and affinity purification as described in Example 4. Thepurified protein or cells transfected with an expression vector encodingthe full length protein can generate monoclonal antibodies against P3G2using conventional techniques, for example those techniques described inU.S. Pat. No. 4,411,993. Briefly BALB-C mice were immunized at 0, 2 and6 weeks with 10 μg P3G2/Fc. The primary immunization was prepared withTITERMAX adjuvant, from Vaxcell, Inc., and subsequent immunization wereprepared with incomplete Freund's adjuvant (IFA). At 11 weeks, the micewere IV boosted with 3-4 μg P3G2 in PBS. Three days after the IV boost,splenocytes were harvested and fused with an Ag8.653 myeloma fusionpartner using 50% aqueous PEG 1500 solution. Hybridoma supernatants werescreened by ELISA using P3G2 transfected COS-1 cells in PBS at 2×10³cells per well and dried to polystyrene 96-well microtiter plates as theplatecoat antigen. Positive supernatants were subsequently confirmed byFACS analysis and RIP using P3G2 transfected COS-1 cells. Hybridomaswere cloned and followed using the same assays. Monoclonal cultures wereexpanded and supernatants purified by affinity chromatography usingBioRad Protein A agarose.

[0129] The monoclonal antibodies to P3G2/Fc were used to screen cellsand cell lines using standard flow cytometry procedures to identifycells on which P3G2 is expressed. Cell lines and cells screened in theflow cytometry analyses were CB23, CB39, RAJI, AK778, K299, PS-1, U937,THP-1, JURKAT and HSB2. For each cell line or cell sample screened, theprocedure involved incubating approximately 100,000 of the cells blockedwith 2% FCS (fetal calf serum), 5% normal Goat serum and 5% rabbit serumin PBS with 5 μg of FITC conjugated mouse anti-P3G2 antibody for 1 hour.Following the incubation the sample was washed 2 times with FACS buffer(2% FCS in PBS). The cells were analyzed for any bound protein usingfluorescent detection flow cytometry to detect FITC. The resultsindicated that LIR-P3G2 antibody binds well to B cell lines CB23 andRAJI1; monocytic cell lines THP-1 and U937; and primary B cell andprimary monocytes. The highest expression of LIR-P3G2 was shown onmonocytes that stained brightly for CD16 and less brightly for CD14 andCD64. The antibody does not bind detectably to T cell lines nor does itbind detectably to primary T cells.

[0130] In a related experiment, the P3G2 antibody generated as describedabove was used in immunoprecipitation experiments. Theimmunoprecipitation analyses involved first surface biotinylating2.5×10⁶ monocytes by washing the cells with PBS and suspending the cellsin a biotinylation buffer of 10 mM sodium borate and 150 mM NaCl at pH8.8, followed by adding 5 μL of a 10 mg/mL solution of biotin-CNHS-ester(D-biotinoyl-e-aminocaproic acid-N-hydroxysuccinimide ester purchasedfrom Amersham) in DMSO to the cells. After quenching the reaction with10 μL of 1 M ammonium chloride per 1 mL of cells and washing the cellsin PBS, the cells were lysed in 1 mL of 0.5% NP40-PBS and the lysate wasrecovered following centrifugation. Then 100 μL of 0.5% NP40-PBS wasadded to 150 μL of the lysate and the resulting mixture was incubatedwith 2 μg/mL of antibody, at 4° C. for 16 hours. Fifty microliters of50% Protein A-Sepharose slurry was added to the antibody mixture and theslurry was shaken at 4° C. for 1 hour. The slurry was centrifuged andthe resulting pellet was washed with 0.75 mL of 0.5% NP40 in PBS sixtimes. Protein bound to the Protein A-Sepharose was eluted with 30 μL ofSDS-PAGE reducing sample buffer and heating at 100° C. for five minutes.

[0131] The eluted proteins were analyzed using 4-20% gradient SDS-PAGEwith enhanced chemiluminescence (ECL) protein markers. Then theelectrophoreses samples were transferred in a Western Blot ontonitrocellulose membranes. The membranes were treated with blockingreagent (0.1% Tween-20 and 3% nonfat dry milk in PBS) for one hour atroom temperature and then they were washed once for 15 minutes followedand twice for 5 minutes with 0.1% Tween-20 in PBS. The washed membraneswere incubated with 10 mL of 1:100 HRP-Streptavidin for 30 minutes andthen washed 1 times for 15 minutes followed by 4 times for 5 minuteswith 0.1% Tween-20 in PBS.

[0132] Bound streptavidin HRP was detected with ECL Detection Reagentspurchased from Amersham and used according to manufacturer'sinstructions. The developed membranes were exposed to x-ray film andthen visualized. The results showed that LIR-P3G2 was immunoprecipitatedfrom CB23 cells and P3G2 transfected COS-1 cells, indicating that P3G2is expressed by these cells.

Example 6 Screening Cells and Cell Lines for Binding to P3G2

[0133] The following describes flow cytometry analyses used to identifycells and cell lines which bind to P3G2. The cells and cell lines testedwere CB23, HSB2, MP-1, Jurkat, primary T cells, primary B cells, andprimary NK cells. For each cell line or cell line tested the procedureinvolved washing the cells three times with FACS buffer (2% FCS in PBSwith 0.2% azide) and incubating each sample (10⁵ cells) in 100 μLblocking buffer (2% FCS, 5% NGS, 5% rabbit serum in PBS) for one hour.For each cell line 4 test samples were prepared, one each having 0, 2,5, or 10 μg of W6/32 (ATCC HB-95) in 100 μL blocking buffer added to thesamples, respectively. W6/32 is an antibody against MHC Class I heavychains (an anti HLA-A, B, and C molecule). Following the addition of theW6/32 solution, the samples were incubated on ice for 1 hour and thenwashed three times with 200 μL of FACS buffer. Then 5 μg of P3G2/Fc inblocking buffer was added to each sample and they were incubated on icefor one hour. The P3G2/Fc competes with W6/32 for binding sites on thecells.

[0134] Following the incubation, the cells were washed three times with200 μL of FACS buffer and treated with mouse anti human Fc/biotin andSAPE for 45 minutes. This treatment causes the anti human Fc/biotin tobind to any cell bound sP3G2/Fc and the SAPE to bind to the anti humanF/Biotin. Since the SAPE is a fluorescing compound its detection usingappropriate excitation and emission conditions positively identifiescell bound P3G2/Fc. Finally the treated cells were washed three timeswith FACS buffer and subjected to flow cytometry to identify cells boundto protein.

[0135] The results demonstrated that W6/32 competed with P3G2 forbinding to all cells and cell lines tested. The P3G2 binding was totallyblocked at 5 μg W6/32 indicating that W6/32 and P3G2 are binding to thesame or overlapping sites on the MHC Class I heavy chains.

Example 7 Screening HSB2 cDNA Library to Isolate a P3G2 Binding Ligand

[0136] The following describes screening a cDNA library from of one ofthe cell lines, HSB-2, a T lymphoblastic leukemia cell line, found tobind P3G2, and identifying a P3G2 binding ligand. An HSB2 cDNA libraryin the mammalian expression vector pDC302, was prepared as generallydescribed in U.S. Pat. No. 5,516,658 and specifically in Kozlosky et al.Oncogene 10.299-306, 1995. Briefly, mRNA was isolated from sorted HSB-2cells and a first cDNA strand was synthesized using 5 μg polyA⁺ and thereverse transcriptase AMV RTase from Life Science. The second cDNAstrand was synthesized using DNA polymerase I from BRL at concentrationof 1.5 U/μL. Using standard techniques as described in Haymerle et al.,Nucl. Acids Res. 14:8615, 1986, the cDNA was ligated into theappropriate site of the pDC302 vector.

[0137]E. coli. strain DH5α cells were transformed with the cDNA libraryin pDC302. After amplifying the library a titer check indicated thatthere was a total of 157,200 clones. The transformed cells were platedinto 15 different plates. Plasmid DNA was isolated from pools consistingof approximately 2,000 clones per pool. The isolated DNA was transfectedinto CV1-EBNA cells (ATCC CRL 10478) using DEAE-dextran followed bychloroquine treatment. The CV1-EBNA cells were maintained in completemedium (Dulbecco's modified Eagles' media containing 10% (v/v) fetalcalf serum, 50 U/mL penicillin, 50 U/mL streptomycin, and 2 mML-glutamine) and were plated to a density of approximately 2×10⁵cells/well in single-well chambered slides. The slides had beenpre-treated with 1 mL of a solution of 10 μg/mL human fibronectin in PBSfor 30 minutes followed by a single washing with PBS. Media was removedfrom adherent cells growing in a layer and replaced with 1.5 mL completemedium containing 66.6 μM chloroquine sulfate. About 0.2 mL of a DNAsolution (2 μg DNA, 0.5 mg/mL DEAE-dextran in complete medium containingchloroquine) was added to the cells and mixture was incubated at 37 Cfor about five hours. Following incubation media was removed and thecells were shocked by adding complete medium containing 10% DMSO for 2.5minutes. After shocking the cells the complete medium was replaced withfresh complete medium and the cells were grown in culture for three daysto permit transient expression of the inserted DNA sequences. Theseconditions led to a 30% to 80% transfection frequency in survivingCV1-EBNA cells.

[0138] Each slide was incubated with 1 mL of P3G2:Fc at a concentrationof 0.45 μg/mL in binding buffer (RPMI 1640 containing 25 mg/mL bovineserum albumin, 2 mg/mL sodium azide, 20 mM Hepes at pH 7.2, and 50 mg/mLnonfat dry milk) at room temperature for 1 hour. After incubating theslides, they were washed with binding buffer and then incubated with Fcspecific ¹²⁵I-mouse anti-human IgG (see Goodwin et al. Cell 73:447-456,1993). This was followed by a second wash with buffer after which theslides were fixed with a 2.5% glutaraldehyde/PBS solution, washed in PBSand allowed to air dry. The slides were dipped in Kodak GTNB-2photographic emulsion (6× dilution in water). After air drying theslides were placed in a dark box and refrigerated. After three days theslides were developed in Kodak D19 developer, rinsed in water and fixedin Agfa G433C fixer. The fixed slides were individually examined under amicroscope at 25-40× magnification. Positive pools demonstrating bindingof sP3G2:Fc were visualized by the presence of autoradiographic silvergrains against the film background. Two positive pools were titered andplated to provide plates containing approximately 200 colonies each.Each plate was scraped to provide pooled plasmid DNA for transfectioninto CV1-EBNA cells and screening as described above. Followingsubsequent breakdowns and screenings, one positive individual colony wasobtained for each pool. The cDNA insert of the positive clones wereidentified as HLA-B44 and HLA-A2, class I MHC antigens.

Example 8 Northern Blot Analysis

[0139] Since the experiments described in Example 4 resulted in thedetection of LIR-P3G2 surface expression on a number of cell lines,conventional Northern Blot analysis procedures were used to study theexpression of LIR-P3G2 and any LIR-P3G2 related mRNAs in differenttissue types. The cell lines selected for Northern Blot analysis wereRAJI, PBT, PBM, YT, HEP3B, HELA, KB, KG-1, IMTLH, HPT, HFF, THP-1, andU937. The following describes the Northern Blot analysis and theanalysis results.

[0140] The cDNA encoding the extracellular region of P3G2 was isolatedusing primers which flank the extracellular region of P3G2 and havingthe following sequences:         Sal I 5′ - TAT GTC GAC CAT GAC CCC CATCCT CAC GGT - 3′ (SEQ ID NO:5)         Bgl II 5′ - TAT AGA TCT ACC CCCAGG TGC CTT CCC AGA CCA (SEQ ID NO:27)

[0141] The PCR template was the full length P3G2 gene isolated asdescribed in Example 3 above. The conditions for the PCR reaction wereas follows: One cycle at 95° C. for 5 minutes; 30 cycles which included95° C. for 45 seconds, 64° C. for 45 seconds and 72° C. for 45 seconds;and, one cycle at 72° C. for 5 minutes. The PCR product was cloned intoPCR II vector, purchased from Invitrogen, in accordance with thesupplier's instructions. The isolated DNA encoding the extracellularregion of P3G2 was used to make a riboprobe with the Ambion MAXISCRIPTKit according to the manufacturer's instructions.

[0142] Northern blots containing poly A+ selected RNA or total RNA froma variety of human cell lines were prepared by resolving RNA samples ona 1.1% agarose-formaldehyde gel, blotting onto Hybond-N as recommendedby the manufacturer (Amersham Corporation) and staining with methyleneblue to monitor RNA concentrations. The blots were prepared using 1 μgof the PolyA+RNA or 10 μg of total RNA and each blot was probed with 10⁶cpm/mL RNA extracellular P3G2 riboprobe, prepared as just described, at63° C. for 16 hours. The probed blots were washed with 2×SSC at 63° C.for 30 minutes 2 times; 1×SSC at 63° C. for 30 minutes 2 times; and,0.1×SSC at 63° C. for 5 minutes 2 times.

[0143] The probed blots were autoradiographically developed. Thedeveloped blots showed that the P3G2 RNA hybridized to a 3.5 kb RNAexpressed by RAJI, CB23 and U937; an approximately 1.5 kb RNA expressedby THP-1; and multiple RNAs ranging from 1.5 kb to 3.5 kb expressed byPBM. These results suggest that different genes having extracellulardomains similar in structure to that of P3G2 may be expressed byperipheral blood monocytes.

Example 9 Probing PBM cDNA Library to Isolate LIR Polypeptides

[0144] The following describes steps taken to screen a peripheral bloodmonocyte cDNA library to isolate polypeptides relating to the P3G2polypeptide using conventional Southern Blot methodologies. A peripheralblood monocyte cDNA library was prepared using substantially the sameprocedures described in Example 7.

[0145] DNA from an initial 15 pools of cDNA having 10,000 clones perpool was digested with Bgl II restriction enzyme and electrophoresed ona 1% agarose gel at 100 V for 2 hours. Southern Blots were prepared byelectroblotting the electrophoresed DNA in 0.55% TBE buffer onto Hybondmembranes. The blotted DNA was denatured in 0.5 M NaOH in 0.6M NaClsolution for 5 minutes and then neutralized in 0.5 M TRIS in 1.5 M NaClat pH 7.8 for 5 minutes. The membranes were placed in a STRATALINKER UVcrosslinker for 20 seconds to crosslink the blotted DNA to the membrane.The membrane and bound DNA were placed in pre-hybridization solution of10× Denhart's Solution, 0.05M TRIS at pH 7.5, 0.9M NaCl, 0.1% sodiumpyrophosphate, 1% SDS and 200 μg/mL salmon sperm DNA at 63° C. for 2hours and then the bound DNA was probed with ³²P labeled probe of DNAencoding the extracellular region of LIR-P3G2, including the signalpeptide and Sal I and Bgl II restriction sites. The concentration of theDNA probe in hybridization solution was 10⁶ CPM per mL of hybridizationsolution. The probed blots were incubated for 16 hours at 63° C. andthen washed with 2×SSC at 63° C. for 1 hour with one solution change; 1×with SSC at 63° C. for one hour with one solution change; and, with0.1×SSC at 68° C. for 45 minutes with one solution change. After dryingthe blots they were autoradiographically developed and visualized forDNA bands which hybridized to the P3G2 extracellular DNA probe.

[0146] The results of the autoradiography visualization indicated thatall pools contained DNA which hybridized to the probe. One pool showing7 positive DNA bands was selected and subsequently subdivided to 10pools having 3,000 clones per pool. Applying subsequent SouthernBlotting methodologies to the 10 pools resulted in one pool showing 9positively hybridizing DNA sequences. Single hybridizing clones wereisolated by standard colony hybridization techniques.

[0147] Duplicate bacterial colonies on filters were probed with the P3G2extracellular probe described above at a concentration of 500,000 cpm/mLat 63° C. for 16 hours. The hybridized filters were washed with 2×SSC at63° C. for 30 minutes, with 1×SSC at 63° C. for 30 minutes; and finallywith 0.1×SSC at 68° C. for 15 minutes.

[0148] Forty-eight clones were visualized as hybridizing on duplicatefilters by autoradiography and DNA obtained from these clones usingstandard DNA preparation methodologies was digested with Bgl II. ThenSouthern Blots of the digests were obtained and probed with the P3G2extracellular probe described above. Seven different sized clonedinserts were identified as positively hybridizing to the P3G2 probe. Thenucleotide sequence of each of the inserts was obtained using automatedsequencing technology. Of the 8 different cloned inserts, one wasidentical in sequence to LIR-P3G2. The others were identified as DNAencoding polypeptides of the new LIR family of polypeptides. Thenucleotide sequences (cDNA) of the isolated LIR family members arepresented in SEQ ID NO:7 (designated pbm25), SEQ ID NO:9 (designatedpbm8), SEQ ID NO:11 (designated pbm36-2), SEQ ID NO:13 (designatedpbm36-4); SEQ ID NO:15 (designated pbmhh); SEQ ID NO:17 (designatedpbm2) and SEQ ID NO:19 (designated pbm17). The amino acid sequencesencoded thereby are presented in SEQ ID NO:8 (designated pbm25), SEQ IDNO:10 (designated pbm8), SEQ ID NO:12 (designated pbm36-2), SEQ ID NO:14(designated pbm36-4), SEQ ID NO:16 (designated pbmhh); SEQ ID NO:18(designated pbm2); and SEQ ID NO:20 (designated pbm17).

Example 10 Screening a Human Dendritic Cell cDNA Library for LIR cDNASequences

[0149] The following describes the isolation and identification of anLIR family member by screening a human bone marrow-derived dendriticcell cDNA library in the λ Zap vector with a radiolabeled Hh0779 cDNAfragment. The Hh0779 cDNA fragment is a 0.7 kb insert of the Hh0779clone previously isolated from a human dendritic cell cDNA library andobtained by restriction digestion with the enzymes PstI and SpeI. TheHh0779 cDNA fragment was labeled with [a-³²P]dCTP using the DECAprime IIDNA labeling kit purchased from Ambion.

[0150] The λ Zap cDNA library was plated at a density of 20,000 pfu perplate to provide a total of 480,000 plagues for the initial screening.The λ Zap cDNA was blotted in duplicate onto Hybond membranes, purchasedfrom Amersham, and then denatured in a solution of 0.5N NaOH and 0.5MNaCl for 5 minutes. The membranes were neutralized in a solution of 0.5MTris (pH 7.8) and 1.5M NaCl for 5 minutes, and then washed in 2×SSC for3 minutes. The cDNA was crosslinked to the Hybond membranes using aSTRATALINKER UV crosslinker in the auto setting.

[0151] The membranes were pre-hybridized at 65° C. for 2.25 hours inhybridization buffer containing 10× Denhardt's, 0.05M Tris (pH 7.5),0.9M NaCl, 0.1% sodium pyrophosphate, 1% SDS and 4 mg/mL heat denaturedsalmon sperm DNA. After the pre-hybridization, the radiolabeled Hh0779cDNA was added to the hybridization buffer to a final concentration of0.54×10⁶ cpm/mL. After 24 hours of hybridization, the membranes werewashed in 0.25×SSC, 0.25% SDS at 65° C. for 1.5 hours. The blots werethen exposed to autoradiographic film to visual positive clones.

[0152] A total of 146 positive clones showing hybridization signals inboth membranes of a duplicate set were identified, isolated, and savedfor future use. Of the 146 clones, 35 were selected for secondaryscreening. The selected clones were plated at low density and singleclones were isolated after hybridization to the HH0779 probe using thehybridization conditions described above. The plasmids were thenisolated from the λ Zap clones using the VCSM13 helper phage purchasedfrom Stratagene. The plasmid DNA was analyzed by restriction digestionand PCR, and the clones containing the 24 largest inserts were selectedand sequenced. Of the 24 sequenced clones, 6 encoded LIR-P3G2, 3 encodedLIR-pbm2, 8 encoded LIR-pbm36-4 and LIR-pbm36-2, 1 encoded LIR-pbm8, 2encoded LIR-pmbhh, and 1 encoded a novel sequence designated LIR-pbmnew.Three clones were identified as encoding amino acid sequences that arenot relevant to the LIR polypeptide family.

Example 11 Association of LIR-P3G2 and Tyrosine Phosphatase SHP-1

[0153] The following describes the tests performed to demonstrate thatLIR-P3G2 and SHP-1 associate. CB23 cells were cultured in RPMI mediumsupplemented with 10% FBS, concentrated by centrifugation and finallysubdivided into two aliquots. One aliquot was stimulated with a solutionof 50 mM/mL sodium pervanadate for 5 minutes. The second aliquot was notstimulated. After stimulation, the cells in each aliquot wereimmediately lysed in RIPA buffer containing 1% NP-40, 0.5% sodiumdeoxycholate, 50 mM Tris pH8, 2 mM EDTA, 0.5 mM sodium orthovanadate, 5mM sodium fluoride, 25 mM β-glycerol phosphate, and protease inhibitors.Samples of 24×10⁶ cell equivalents were incubated for 2 hours at 4° C.with either 5 μg/mL of anti-SHP-1 antibody purchased from TransductionLaboratories, or 5 μg/mL of an isotype-matched antibody control(anti-Flag-M5 IgG1). The resulting immunocomplexes were precipitated byincubation with protein G-agarose (Boehringer Mannheim), washed, andresuspended in 40 mL of 2× SDS-PAGE sample buffer. Twenty microliters ofeach immunoprecipitate were loaded onto electrophoresis gels,electrophoresed under reducing conditions, and transferred tonitrocellulose membranes purchased from Amersham. Western blots wereprobed with anti-LIR-P3G2 polyclonal antisera and the immunocomplexeswere detected by enhanced chemiluminescence (NEN).

[0154] A protein having a molecular weight of approximately 120 kDa andcorresponding to LIR-P3G2 was readily detected in SHP-1immunoprecipitates, but not in anti-Flag-M5 antibody. The LIR-P3G2 bandwas not seen in the absence of sodium pervanadate treatment, showingthat tyrosine phosphorylation of LIR-P3G2 is essential for theassociation of LIR-1 and SHP-1. This data demonstrates that LIR-P3G2, byanalogy with molecules possesses the ITIM motif, sends an inhibitorysignal intracellularly when it interacts with its counterstructures,viral or cellular MHC class I molecules.

Example 12 Generating Antibodies Immunoreactive with LIR Polypeptides

[0155] The following describes generating monoclonal antibodyimmunoreactive with LIR family members. A purified LIR polypeptide isprepared by COS-1 cell expression and affinity purification as describedin Example 4. The purified protein or cells transfected with anexpression vector encoding the full length protein can generatemonoclonal antibodies against the LIR polypeptide using conventionaltechniques, for example those techniques described in U.S. Pat. No.4,411,993. Briefly BALB-C mice are immunized at 0, 2 and 6 weeks with 10μg of the LIR polypeptide. The primary immunization is prepared withTITERMAX adjuvant and subsequent immunizations are prepared withincomplete Freund's adjuvant (IFA). At 11 weeks, the mice are IV boostedwith 3-4 μg the LIR polypeptide in PBS. Three days after the IV boost,splenocytes are harvested and fused with an Ag8.653 myeloma fusionpartner using 50% aqueous PEG 1500 solution. Hybridoma supernatants arescreened by ELISA using the LIR transfected cells in PBS at 7×10³ cellsper well and dried to polystyrene 96-well microtiter plates as theplatecoat antigen. Positive supernatants are subsequently confirmed byFACS analysis and RIP using LIR transfected cells. Hybridomas are clonedand followed in the same manner of screening. Monoclonal cultures areexpanded and supernatants purified by affinity chromatography.

1 29 2922 base pairs nucleic acid single linear cDNA NO LeukocyteImmunoglobulin Receptor P3G2 CDS 310..2259 1 AGGGCCACGC GTGCATGCGTCGACTGGAAC GAGACGACCT GCTGTGACCC CCTTGTGGGC 60 ACTCCATTGG TTTTATGGCGCCTCTACTTT CTGGAGTTTG TGTAAAACAA AAATATTATG 120 GTCTTTGTGC ACATTTACATCAAGCTCAGC CTGGGCGGCA CAGCCAGATG CGAGATGCGT 180 CTCTGCTGAT CTGAGTCTGCCTGCAGCATG GACCTGGGTC TTCCCTGAAG CATCTCCAGG 240 GCTGGAGGGA CGACTGCCATGCACCGAGGG CTCATCCATC CACAGAGCAG GGCAGTGGGA 300 GGAGACGCC ATG ACC CCCATC CTC ACG GTC CTG ATC TGT CTC GGG CTG 348 Met Thr Pro Ile Leu Thr ValLeu Ile Cys Leu Gly Leu 1 5 10 AGT CTG GGC CCC CGG ACC CAC GTG CAG GCAGGG CAC CTC CCC AAG CCC 396 Ser Leu Gly Pro Arg Thr His Val Gln Ala GlyHis Leu Pro Lys Pro 15 20 25 ACC CTC TGG GCT GAA CCA GGC TCT GTG ATC ACCCAG GGG AGT CCT GTG 444 Thr Leu Trp Ala Glu Pro Gly Ser Val Ile Thr GlnGly Ser Pro Val 30 35 40 45 ACC CTC AGG TGT CAG GGG GGC CAG GAG ACC CAGGAG TAC CGT CTA TAT 492 Thr Leu Arg Cys Gln Gly Gly Gln Glu Thr Gln GluTyr Arg Leu Tyr 50 55 60 AGA GAA AAG AAA ACA GCA CCC TGG ATT ACA CGG ATCCCA CAG GAG CTT 540 Arg Glu Lys Lys Thr Ala Pro Trp Ile Thr Arg Ile ProGln Glu Leu 65 70 75 GTG AAG AAG GGC CAG TTC CCC ATC CCA TCC ATC ACC TGGGAA CAT GCA 588 Val Lys Lys Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp GluHis Ala 80 85 90 GGG CGG TAT CGC TGT TAC TAT GGT AGC GAC ACT GCA GGC CGCTCA GAG 636 Gly Arg Tyr Arg Cys Tyr Tyr Gly Ser Asp Thr Ala Gly Arg SerGlu 95 100 105 AGC AGT GAC CCC CTG GAG CTG GTG GTG ACA GGA GCC TAC ATCAAA CCC 684 Ser Ser Asp Pro Leu Glu Leu Val Val Thr Gly Ala Tyr Ile LysPro 110 115 120 125 ACC CTC TCA GCC CAG CCC AGC CCC GTG GTG AAC TCA GGAGGG AAT GTA 732 Thr Leu Ser Ala Gln Pro Ser Pro Val Val Asn Ser Gly GlyAsn Val 130 135 140 ACC CTC CAG TGT GAC TCA CAG GTG GCA TTT GAT GGC TTCATT CTG TGT 780 Thr Leu Gln Cys Asp Ser Gln Val Ala Phe Asp Gly Phe IleLeu Cys 145 150 155 AAG GAA GGA GAA GAT GAA CAC CCA CAA TGC CTG AAC TCCCAG CCC CAT 828 Lys Glu Gly Glu Asp Glu His Pro Gln Cys Leu Asn Ser GlnPro His 160 165 170 GCC CGT GGG TCG TCC CGC GCC ATC TTC TCC GTG GGC CCCGTG AGC CCG 876 Ala Arg Gly Ser Ser Arg Ala Ile Phe Ser Val Gly Pro ValSer Pro 175 180 185 AGT CGC AGG TGG TGG TAC AGG TGC TAT GCT TAT GAC TCGAAC TCT CCC 924 Ser Arg Arg Trp Trp Tyr Arg Cys Tyr Ala Tyr Asp Ser AsnSer Pro 190 195 200 205 TAT GAG TGG TCT CTA CCC AGT GAT CTC CTG GAG CTCCTG GTC CTA GGT 972 Tyr Glu Trp Ser Leu Pro Ser Asp Leu Leu Glu Leu LeuVal Leu Gly 210 215 220 GTT TCT AAG AAG CCA TCA CTC TCA GTG CAG CCA GGTCCT ATC GTG GCC 1020 Val Ser Lys Lys Pro Ser Leu Ser Val Gln Pro Gly ProIle Val Ala 225 230 235 CCT GAG GAG ACC CTG ACT CTG CAG TGT GGC TCT GATGCT GGC TAC AAC 1068 Pro Glu Glu Thr Leu Thr Leu Gln Cys Gly Ser Asp AlaGly Tyr Asn 240 245 250 AGA TTT GTT CTG TAT AAG GAC GGG GAA CGT GAC TTCCTT CAG CTC GCT 1116 Arg Phe Val Leu Tyr Lys Asp Gly Glu Arg Asp Phe LeuGln Leu Ala 255 260 265 GGC GCA CAG CCC CAG GCT GGG CTC TCC CAG GCC AACTTC ACC CTG GGC 1164 Gly Ala Gln Pro Gln Ala Gly Leu Ser Gln Ala Asn PheThr Leu Gly 270 275 280 285 CCT GTG AGC CGC TCC TAC GGG GGC CAG TAC AGATGC TAC GGT GCA CAC 1212 Pro Val Ser Arg Ser Tyr Gly Gly Gln Tyr Arg CysTyr Gly Ala His 290 295 300 AAC CTC TCC TCC GAG TGG TCG GCC CCC AGC GACCCC CTG GAC ATC CTG 1260 Asn Leu Ser Ser Glu Trp Ser Ala Pro Ser Asp ProLeu Asp Ile Leu 305 310 315 ATC GCA GGA CAG TTC TAT GAC AGA GTC TCC CTCTCG GTG CAG CCG GGC 1308 Ile Ala Gly Gln Phe Tyr Asp Arg Val Ser Leu SerVal Gln Pro Gly 320 325 330 CCC ACG GTG GCC TCA GGA GAG AAC GTG ACC CTGCTG TGT CAG TCA CAG 1356 Pro Thr Val Ala Ser Gly Glu Asn Val Thr Leu LeuCys Gln Ser Gln 335 340 345 GGA TGG ATG CAA ACT TTC CTT CTG ACC AAG GAGGGG GCA GCT GAT GAC 1404 Gly Trp Met Gln Thr Phe Leu Leu Thr Lys Glu GlyAla Ala Asp Asp 350 355 360 365 CCA TGG CGT CTA AGA TCA ACG TAC CAA TCTCAA AAA TAC CAG GCT GAA 1452 Pro Trp Arg Leu Arg Ser Thr Tyr Gln Ser GlnLys Tyr Gln Ala Glu 370 375 380 TTC CCC ATG GGT CCT GTG ACC TCA GCC CATGCG GGG ACC TAC AGG TGC 1500 Phe Pro Met Gly Pro Val Thr Ser Ala His AlaGly Thr Tyr Arg Cys 385 390 395 TAC GGC TCA CAG AGC TCC AAA CCC TAC CTGCTG ACT CAC CCC AGT GAC 1548 Tyr Gly Ser Gln Ser Ser Lys Pro Tyr Leu LeuThr His Pro Ser Asp 400 405 410 CCC CTG GAG CTC GTG GTC TCA GGA CCG TCTGGG GGC CCC AGC TCC CCG 1596 Pro Leu Glu Leu Val Val Ser Gly Pro Ser GlyGly Pro Ser Ser Pro 415 420 425 ACA ACA GGC CCC ACC TCC ACA TCT GGC CCTGAG GAC CAG CCC CTC ACC 1644 Thr Thr Gly Pro Thr Ser Thr Ser Gly Pro GluAsp Gln Pro Leu Thr 430 435 440 445 CCC ACC GGG TCG GAT CCC CAG AGT GGTCTG GGA AGG CAC CTG GGG GTT 1692 Pro Thr Gly Ser Asp Pro Gln Ser Gly LeuGly Arg His Leu Gly Val 450 455 460 GTG ATC GGC ATC TTG GTG GCC GTC ATCCTA CTG CTC CTC CTC CTC CTC 1740 Val Ile Gly Ile Leu Val Ala Val Ile LeuLeu Leu Leu Leu Leu Leu 465 470 475 CTC CTC TTC CTC ATC CTC CGA CAT CGACGT CAG GGC AAA CAC TGG ACA 1788 Leu Leu Phe Leu Ile Leu Arg His Arg ArgGln Gly Lys His Trp Thr 480 485 490 TCG ACC CAG AGA AAG GCT GAT TTC CAACAT CCT GCA GGG GCT GTG GGG 1836 Ser Thr Gln Arg Lys Ala Asp Phe Gln HisPro Ala Gly Ala Val Gly 495 500 505 CCA GAG CCC ACA GAC AGA GGC CTG CAGTGG AGG TCC AGC CCA GCT GCC 1884 Pro Glu Pro Thr Asp Arg Gly Leu Gln TrpArg Ser Ser Pro Ala Ala 510 515 520 525 GAT GCC CAG GAA GAA AAC CTC TATGCT GCC GTG AAG CAC ACA CAG CCT 1932 Asp Ala Gln Glu Glu Asn Leu Tyr AlaAla Val Lys His Thr Gln Pro 530 535 540 GAG GAT GGG GTG GAG ATG GAC ACTCGG AGC CCA CAC GAT GAA GAC CCC 1980 Glu Asp Gly Val Glu Met Asp Thr ArgSer Pro His Asp Glu Asp Pro 545 550 555 CAG GCA GTG ACG TAT GCC GAG GTGAAA CAC TCC AGA CCT AGG AGA GAA 2028 Gln Ala Val Thr Tyr Ala Glu Val LysHis Ser Arg Pro Arg Arg Glu 560 565 570 ATG GCC TCT CCT CCT TCC CCA CTGTCT GGG GAA TTC CTG GAC ACA AAG 2076 Met Ala Ser Pro Pro Ser Pro Leu SerGly Glu Phe Leu Asp Thr Lys 575 580 585 GAC AGA CAG GCG GAA GAG GAC AGGCAG ATG GAC ACT GAG GCT GCT GCA 2124 Asp Arg Gln Ala Glu Glu Asp Arg GlnMet Asp Thr Glu Ala Ala Ala 590 595 600 605 TCT GAA GCC CCC CAG GAT GTGACC TAC GCC CAG CTG CAC AGC TTG ACC 2172 Ser Glu Ala Pro Gln Asp Val ThrTyr Ala Gln Leu His Ser Leu Thr 610 615 620 CTT AGA CGG AAG GCA ACT GAGCCT CCT CCA TCC CAG GAA GGG CCC TCT 2220 Leu Arg Arg Lys Ala Thr Glu ProPro Pro Ser Gln Glu Gly Pro Ser 625 630 635 CCA GCT GTG CCC AGC ATC TACGCC ACT CTG GCC ATC CAC TAG 2262 Pro Ala Val Pro Ser Ile Tyr Ala Thr LeuAla Ile His 640 645 650 CCCAGGGGGG GACGCAGACC CCACACTCCA TGGAGTCTGGAATGCATGGG AGCTGCCCCC 2322 CCAGTGGACA CCATTGGACC CCACCCAGCC TGGATCTACCCCAGGAGACT CTGGGAACTT 2382 TTAGGGGTCA CTCAATTCTG CAGTATAAAT AACTAATGTCTCTACAATTT TGAAATAAAG 2442 CAACAGACTT CTCAATAATC AATGAAGTAG CTGAGAAAACTAAGTCAGAA AGTGCATTAA 2502 ACTGAATCAC AATGTAAATA TTACACATCA AGCGATGAAACTGGAAAACT ACAAGCCACG 2562 AATGAATGAA TTAGGAAAGA AAAAAAGTAG GAAATGAATGATCTTGGCTT TCCTATAAGA 2622 AATTTAGGGC AGGGCACGGT GGCTCACGCC TGTAATTCCAGCACTTTGGG AGGCCGAGGC 2682 GGGCAGATCA CGAGTTCAGG AGATCGAGAC CATCTTGGCCAACATGGTGA AACCCTGTCT 2742 CTCCTAAAAA TACAAAAATT AGCTGGATGT GGTGGCAGTGCCTGTAATCC CAGCTATTTG 2802 GGAGGCTGAG GCAGGAGAAT CGCTTGAACC AGGGAGTCAGAGGTTTCAGT GAGCCAAGAT 2862 CGCACCACTG CTCTCCAGCC TGGCGACAAG CAGGTCGTCTCGTTCCAGTC GACGGCCCAT 2922 650 amino acids amino acid linear protein 2Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 1015 Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp 20 2530 Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Arg 35 4045 Cys Gln Gly Gly Gln Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys 50 5560 Lys Thr Ala Pro Trp Ile Thr Arg Ile Pro Gln Glu Leu Val Lys Lys 65 7075 80 Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr 8590 95 Arg Cys Tyr Tyr Gly Ser Asp Thr Ala Gly Arg Ser Glu Ser Ser Asp100 105 110 Pro Leu Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr LeuSer 115 120 125 Ala Gln Pro Ser Pro Val Val Asn Ser Gly Gly Asn Val ThrLeu Gln 130 135 140 Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ile Leu CysLys Glu Gly 145 150 155 160 Glu Asp Glu His Pro Gln Cys Leu Asn Ser GlnPro His Ala Arg Gly 165 170 175 Ser Ser Arg Ala Ile Phe Ser Val Gly ProVal Ser Pro Ser Arg Arg 180 185 190 Trp Trp Tyr Arg Cys Tyr Ala Tyr AspSer Asn Ser Pro Tyr Glu Trp 195 200 205 Ser Leu Pro Ser Asp Leu Leu GluLeu Leu Val Leu Gly Val Ser Lys 210 215 220 Lys Pro Ser Leu Ser Val GlnPro Gly Pro Ile Val Ala Pro Glu Glu 225 230 235 240 Thr Leu Thr Leu GlnCys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val 245 250 255 Leu Tyr Lys AspGly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln 260 265 270 Pro Gln AlaGly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser 275 280 285 Arg SerTyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser 290 295 300 SerGlu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly 305 310 315320 Gln Phe Tyr Asp Arg Val Ser Leu Ser Val Gln Pro Gly Pro Thr Val 325330 335 Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp Met340 345 350 Gln Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala Asp Asp Pro TrpArg 355 360 365 Leu Arg Ser Thr Tyr Gln Ser Gln Lys Tyr Gln Ala Glu PhePro Met 370 375 380 Gly Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg CysTyr Gly Ser 385 390 395 400 Gln Ser Ser Lys Pro Tyr Leu Leu Thr His ProSer Asp Pro Leu Glu 405 410 415 Leu Val Val Ser Gly Pro Ser Gly Gly ProSer Ser Pro Thr Thr Gly 420 425 430 Pro Thr Ser Thr Ser Gly Pro Glu AspGln Pro Leu Thr Pro Thr Gly 435 440 445 Ser Asp Pro Gln Ser Gly Leu GlyArg His Leu Gly Val Val Ile Gly 450 455 460 Ile Leu Val Ala Val Ile LeuLeu Leu Leu Leu Leu Leu Leu Leu Phe 465 470 475 480 Leu Ile Leu Arg HisArg Arg Gln Gly Lys His Trp Thr Ser Thr Gln 485 490 495 Arg Lys Ala AspPhe Gln His Pro Ala Gly Ala Val Gly Pro Glu Pro 500 505 510 Thr Asp ArgGly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala Gln 515 520 525 Glu GluAsn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp Gly 530 535 540 ValGlu Met Asp Thr Arg Ser Pro His Asp Glu Asp Pro Gln Ala Val 545 550 555560 Thr Tyr Ala Glu Val Lys His Ser Arg Pro Arg Arg Glu Met Ala Ser 565570 575 Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln580 585 590 Ala Glu Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser GluAla 595 600 605 Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu Thr LeuArg Arg 610 615 620 Lys Ala Thr Glu Pro Pro Pro Ser Gln Glu Gly Pro SerPro Ala Val 625 630 635 640 Pro Ser Ile Tyr Ala Thr Leu Ala Ile His 645650 2777 base pairs nucleic acid single linear cDNA 18a3 CDS 168..2123 3AGCTCAGCCT GGGCGGCACA GCCAGATGCG AGATGCGTCT CTGCTGATCT GAGTCTGCCT 60GCAGCATGGA CCTGGGTCTT CCCTGAAGCA TCTCCAGGGC TGGAGGGACG ACTGCCATGC 120ACCGAGGGCT CATCCATCCA CAGAGCAGGG CAGTGGGAGG AGACGCC ATG ACC CCC 176 MetThr Pro 1 ATC CTC ACG GTC CTG ATC TGT CTC GGG CTG AGT CTG GGC CCC AGGACC 224 Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly Pro Arg Thr5 10 15 CAC GTG CAG GCA GGG CAC CTC CCC AAG CCC ACC CTC TGG GCT GAA CCA272 His Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp Ala Glu Pro 2025 30 35 GGC TCT GTG ATC ACC CAG GGG AGT CCT GTG ACC CTC AGG TGT CAG GGG320 Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Arg Cys Gln Gly 4045 50 GGC CAG GAG ACC CAG GAG TAC CGT CTA TAT AGA GAA AAG AAA ACA GCA368 Gly Gln Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys Lys Thr Ala 5560 65 CTC TGG ATT ACA CGG ATC CCA CAG GAG CTT GTG AAG AAG GGC CAG TTC416 Leu Trp Ile Thr Arg Ile Pro Gln Glu Leu Val Lys Lys Gly Gln Phe 7075 80 CCC ATC CCA TCC ATC ACC TGG GAA CAT GCA GGG CGG TAT CGC TGT TAC464 Pro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr Arg Cys Tyr 8590 95 TAT GGT AGC GAC ACT GCA GGC CGC TCA GAG AGC AGT GAC CCC CTG GAG512 Tyr Gly Ser Asp Thr Ala Gly Arg Ser Glu Ser Ser Asp Pro Leu Glu 100105 110 115 CTG GTG GTG ACA GGA GCC TAC ATC AAA CCC ACC CTC TCA GCC CAGCCC 560 Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser Ala Gln Pro120 125 130 AGC CCC GTG GTG AAC TCA GGA GGG AAT GTA ATC CTC CAG TGT GACTCA 608 Ser Pro Val Val Asn Ser Gly Gly Asn Val Ile Leu Gln Cys Asp Ser135 140 145 CAG GTG GCA TTT GAT GGC TTC AGT CTG TGT AAG GAA GGA GAA GATGAA 656 Gln Val Ala Phe Asp Gly Phe Ser Leu Cys Lys Glu Gly Glu Asp Glu150 155 160 CAC CCA CAA TGC CTG AAC TCC CAG CCC CAT GCC CGT GGG TCG TCCCGC 704 His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly Ser Ser Arg165 170 175 GCC ATC TTC TCC GTG GGC CCC GTG AGC CCG AGT CGC AGG TGG TGGTAC 752 Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg Trp Trp Tyr180 185 190 195 AGG TGC TAT GCT TAT GAC TCG AAC TCT CCC TAT GAG TGG TCTCTA CCC 800 Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr Glu Trp Ser LeuPro 200 205 210 AGT GAT CTC CTG GAG CTC CTG GTC CTA GGT GTT TCT AAG AAGCCA TCA 848 Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val Ser Lys Lys ProSer 215 220 225 CTC TCA GTG CAG CCA GGT CCT ATC GTG GCC CCT GAG GAG ACCCTG ACT 896 Leu Ser Val Gln Pro Gly Pro Ile Val Ala Pro Glu Glu Thr LeuThr 230 235 240 CTG CAG TGT GGC TCT GAT GCT GGC TAC AAC AGA TTT GTT CTGTAT AAG 944 Leu Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val Leu TyrLys 245 250 255 GAC GGG GAA CGT GAC TTC CTT CAG CTC GCT GGC GCA CAG CCCCAG GCT 992 Asp Gly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln Pro GlnAla 260 265 270 275 GGG CTC TCC CAG GCC AAC TTC ACC CTG GGC CCT GTG AGCCGC TCC TAC 1040 Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser ArgSer Tyr 280 285 290 GGG GGC CAG TAC AGA TGC TAC GGT GCA CAC AAC CTC TCCTCC GAG TGG 1088 Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser SerGlu Trp 295 300 305 TCG GCC CCC AGT GAC CCC CTG GAC ATC CTG ATC GCA GGACAG TTC TAT 1136 Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly GlnPhe Tyr 310 315 320 GAC AGA GTC TCC CTC TCG GTG CAG CCG GGC CCC ACG GTGGCC TCA GGA 1184 Asp Arg Val Ser Leu Ser Val Gln Pro Gly Pro Thr Val AlaSer Gly 325 330 335 GAG AAC GTG ACC CTG CTG TGT CAG TCA CAG GGA TGG ATGCAA ACT TTC 1232 Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Trp Met GlnThr Phe 340 345 350 355 CTT CTG ACC AAG GAG GGG GCA GCT GAT GAC CCA TGGCGT CTA AGA TCA 1280 Leu Leu Thr Lys Glu Gly Ala Ala Asp Asp Pro Trp ArgLeu Arg Ser 360 365 370 ACG TAC CAA TCT CAA AAA TAC CAG GCT GAA TTC CCCATG GGT CCT GTG 1328 Thr Tyr Gln Ser Gln Lys Tyr Gln Ala Glu Phe Pro MetGly Pro Val 375 380 385 ACC TCA GCC CAT GCG GGG ACC TAC AGG TGC TAC GGCTCA CAG AGC TCC 1376 Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly SerGln Ser Ser 390 395 400 AAA CCC TAC CTG CTG ACT CAC CCC AGT GAC CCC CTGGAG CTC GTG GTC 1424 Lys Pro Tyr Leu Leu Thr His Pro Ser Asp Pro Leu GluLeu Val Val 405 410 415 TCA GGA CCG TCT GGG GGC CCC AGC TCC CCG ACA ACAGGC CCC ACC TCC 1472 Ser Gly Pro Ser Gly Gly Pro Ser Ser Pro Thr Thr GlyPro Thr Ser 420 425 430 435 ACA TCT GCA GGC CCT GAG GAC CAG CCC CTC ACCCCC ACC GGG TCG GAT 1520 Thr Ser Ala Gly Pro Glu Asp Gln Pro Leu Thr ProThr Gly Ser Asp 440 445 450 CCC CAG AGT GGT CTG GGA AGG CAC CTG GGG GTTGTG ATC GGC ATC TTG 1568 Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val ValIle Gly Ile Leu 455 460 465 GTG GCC GTC ATC CTA CTG CTC CTC CTC CTC CTCCTC CTC TTC CTC ATC 1616 Val Ala Val Ile Leu Leu Leu Leu Leu Leu Leu LeuLeu Phe Leu Ile 470 475 480 CTC CGA CAT CGA CGT CAG GGC AAA CAC TGG ACATCG ACC CAG AGA AAG 1664 Leu Arg His Arg Arg Gln Gly Lys His Trp Thr SerThr Gln Arg Lys 485 490 495 GCT GAT TTC CAA CAT CCT GCA GGG GCT GTG GGGCCA GAG CCC ACA GAC 1712 Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly ProGlu Pro Thr Asp 500 505 510 515 AGA GGC CTG CAG TGG AGG TCC AGC CCA GCTGCC GAT GCC CAG GAA GAA 1760 Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala AlaAsp Ala Gln Glu Glu 520 525 530 AAC CTC TAT GCT GCC GTG AAG CAC ACA CAGCCT GAG GAT GGG GTG GAG 1808 Asn Leu Tyr Ala Ala Val Lys His Thr Gln ProGlu Asp Gly Val Glu 535 540 545 ATG GAC ACT CGG CAG AGC CCA CAC GAT GAAGAC CCC CAG GCA GTG ACG 1856 Met Asp Thr Arg Gln Ser Pro His Asp Glu AspPro Gln Ala Val Thr 550 555 560 TAT GCC GAG GTG AAA CAC TCC AGA CCT AGGAGA GAA ATG GCC TCT CCT 1904 Tyr Ala Glu Val Lys His Ser Arg Pro Arg ArgGlu Met Ala Ser Pro 565 570 575 CCT TCC CCA CTG TCT GGG GAA TTC CTG GACACA AAG GAC AGA CAG GCG 1952 Pro Ser Pro Leu Ser Gly Glu Phe Leu Asp ThrLys Asp Arg Gln Ala 580 585 590 595 GAA GAG GAC AGG CAG ATG GAC ACT GAGGCT GCT GCA TCT GAA GCC CCC 2000 Glu Glu Asp Arg Gln Met Asp Thr Glu AlaAla Ala Ser Glu Ala Pro 600 605 610 CAG GAT GTG ACC TAC GCC CAG CTG CACAGC TTG ACC CTC AGA CGG GAG 2048 Gln Asp Val Thr Tyr Ala Gln Leu His SerLeu Thr Leu Arg Arg Glu 615 620 625 GCA ACT GAG CCT CCT CCA TCC CAG GAAGGG CCC TCT CCA GCT GTG CCC 2096 Ala Thr Glu Pro Pro Pro Ser Gln Glu GlyPro Ser Pro Ala Val Pro 630 635 640 AGC ATC TAC GCC ACT CTG GCC ATC CACTAG CCCAGGGGGG GACGCAGACC 2146 Ser Ile Tyr Ala Thr Leu Ala Ile His 645650 CCACACTCCA TGGAGTCTGG AATGCATGGG AGCTGCCCCC CCAGTGGACA CCATTGGACC2206 CCACCCAGCC TGGATCTACC CCAGGAGACT CTGGGAACTT TTAGGGGTCA CTCAATTCTG2266 CAGTATAAAT AACTAATGTC TCTACAATTT TGAAATAAAG CAATAGACTT CTCAATAATC2326 AATGAAGTAG CTGAGAAAAC TAAGTCAGAA AGTGCATTAA ACTGAATCAC AATGTAAATA2386 TTACACATCA AGCGATGAAA CTGGAAAACT ACAAGCCACG AATGAATGAA TTAGGAAAGA2446 AAAAAAGTAG GAAATGAATG ATCTTGGCTT TCCTATAAGA AATTTAGGGC AGGGCACGGT2506 GGCTCACGCC TGTAATTCCA GCACTTTGGG AGGCCGAGGC GGGCAGATCA CGAGTTCAGG2566 AGATCGAGAC CATCTTGGCC AACATGGTGA AACCCTGTCT CTCCTAAAAA TACAAAAATT2626 AGCTGGATGT GGTGGCAGTG CCTGTAATCC CAGCTATTTG GGAGGCTGAG GCAGGAGAAT2686 CGCTTGAACC AGGGAGTCAG AGGTTTCAGT GAGCCAAGAT CGCACCACTG CTCTCCAGCC2746 TGGCGACAGA GGGAGACTCC ATCTCAAATT A 2777 652 amino acids amino acidlinear protein 4 Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu SerLeu Gly 1 5 10 15 Pro Arg Thr His Val Gln Ala Gly His Leu Pro Lys ProThr Leu Trp 20 25 30 Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro ValThr Leu Arg 35 40 45 Cys Gln Gly Gly Gln Glu Thr Gln Glu Tyr Arg Leu TyrArg Glu Lys 50 55 60 Lys Thr Ala Leu Trp Ile Thr Arg Ile Pro Gln Glu LeuVal Lys Lys 65 70 75 80 Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu HisAla Gly Arg Tyr 85 90 95 Arg Cys Tyr Tyr Gly Ser Asp Thr Ala Gly Arg SerGlu Ser Ser Asp 100 105 110 Pro Leu Glu Leu Val Val Thr Gly Ala Tyr IleLys Pro Thr Leu Ser 115 120 125 Ala Gln Pro Ser Pro Val Val Asn Ser GlyGly Asn Val Ile Leu Gln 130 135 140 Cys Asp Ser Gln Val Ala Phe Asp GlyPhe Ser Leu Cys Lys Glu Gly 145 150 155 160 Glu Asp Glu His Pro Gln CysLeu Asn Ser Gln Pro His Ala Arg Gly 165 170 175 Ser Ser Arg Ala Ile PheSer Val Gly Pro Val Ser Pro Ser Arg Arg 180 185 190 Trp Trp Tyr Arg CysTyr Ala Tyr Asp Ser Asn Ser Pro Tyr Glu Trp 195 200 205 Ser Leu Pro SerAsp Leu Leu Glu Leu Leu Val Leu Gly Val Ser Lys 210 215 220 Lys Pro SerLeu Ser Val Gln Pro Gly Pro Ile Val Ala Pro Glu Glu 225 230 235 240 ThrLeu Thr Leu Gln Cys Gly Ser Asp Ala Gly Tyr Asn Arg Phe Val 245 250 255Leu Tyr Lys Asp Gly Glu Arg Asp Phe Leu Gln Leu Ala Gly Ala Gln 260 265270 Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser 275280 285 Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu Ser290 295 300 Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile AlaGly 305 310 315 320 Gln Phe Tyr Asp Arg Val Ser Leu Ser Val Gln Pro GlyPro Thr Val 325 330 335 Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln SerGln Gly Trp Met 340 345 350 Gln Thr Phe Leu Leu Thr Lys Glu Gly Ala AlaAsp Asp Pro Trp Arg 355 360 365 Leu Arg Ser Thr Tyr Gln Ser Gln Lys TyrGln Ala Glu Phe Pro Met 370 375 380 Gly Pro Val Thr Ser Ala His Ala GlyThr Tyr Arg Cys Tyr Gly Ser 385 390 395 400 Gln Ser Ser Lys Pro Tyr LeuLeu Thr His Pro Ser Asp Pro Leu Glu 405 410 415 Leu Val Val Ser Gly ProSer Gly Gly Pro Ser Ser Pro Thr Thr Gly 420 425 430 Pro Thr Ser Thr SerAla Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr 435 440 445 Gly Ser Asp ProGln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile 450 455 460 Gly Ile LeuVal Ala Val Ile Leu Leu Leu Leu Leu Leu Leu Leu Leu 465 470 475 480 PheLeu Ile Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr 485 490 495Gln Arg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu 500 505510 Pro Thr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala 515520 525 Gln Glu Glu Asn Leu Tyr Ala Ala Val Lys His Thr Gln Pro Glu Asp530 535 540 Gly Val Glu Met Asp Thr Arg Gln Ser Pro His Asp Glu Asp ProGln 545 550 555 560 Ala Val Thr Tyr Ala Glu Val Lys His Ser Arg Pro ArgArg Glu Met 565 570 575 Ala Ser Pro Pro Ser Pro Leu Ser Gly Glu Phe LeuAsp Thr Lys Asp 580 585 590 Arg Gln Ala Glu Glu Asp Arg Gln Met Asp ThrGlu Ala Ala Ala Ser 595 600 605 Glu Ala Pro Gln Asp Val Thr Tyr Ala GlnLeu His Ser Leu Thr Leu 610 615 620 Arg Arg Glu Ala Thr Glu Pro Pro ProSer Gln Glu Gly Pro Ser Pro 625 630 635 640 Ala Val Pro Ser Ile Tyr AlaThr Leu Ala Ile His 645 650 30 nucleotides nucleic acid single linearOligonucleotide 5 TATGTCGACC ATGACCCCCA TCCTCACGGT 30 52 nucleotidesnucleic acid single linear Oligonucleotide 6 TATGGGCTCT GCTCCAGGAGAAGATCTTCC TTCTATAACC CCCAGGTGCC TT 52 1605 base pairs nucleic acidsingle linear cDNA pbm25 CDS 93..1409 7 GAGCCTCCAA GTGTCCACAC CCTGTGTGTCCTCTGTCCTG CCAGCACCGA GGGCTCATCC 60 ATCCACAGAG CAGTGCAGTG GGAGGAGACG CCATG ACC CCC ATC CTC ACG GTC 113 Met Thr Pro Ile Leu Thr Val 1 5 CTG ATCTGT CTC GGG CTG AGC CTG GAC CCC AGG ACC CAC GTG CAG GCA 161 Leu Ile CysLeu Gly Leu Ser Leu Asp Pro Arg Thr His Val Gln Ala 10 15 20 GGG CCC CTCCCC AAG CCC ACC CTC TGG GCT GAG CCA GGC TCT GTG ATC 209 Gly Pro Leu ProLys Pro Thr Leu Trp Ala Glu Pro Gly Ser Val Ile 25 30 35 ACC CAA GGG AGTCCT GTG ACC CTC AGG TGT CAG GGG AGC CTG GAG ACG 257 Thr Gln Gly Ser ProVal Thr Leu Arg Cys Gln Gly Ser Leu Glu Thr 40 45 50 55 CAG GAG TAC CATCTA TAT AGA GAA AAG AAA ACA GCA CTC TGG ATT ACA 305 Gln Glu Tyr His LeuTyr Arg Glu Lys Lys Thr Ala Leu Trp Ile Thr 60 65 70 CGG ATC CCA CAG GAGCTT GTG AAG AAG GGC CAG TTC CCC ATC CTA TCC 353 Arg Ile Pro Gln Glu LeuVal Lys Lys Gly Gln Phe Pro Ile Leu Ser 75 80 85 ATC ACC TGG GAA CAT GCAGGG CGG TAT TGC TGT ATC TAT GGC AGC CAC 401 Ile Thr Trp Glu His Ala GlyArg Tyr Cys Cys Ile Tyr Gly Ser His 90 95 100 ACT GCA GGC CTC TCA GAGAGC AGT GAC CCC CTG GAG CTG GTG GTG ACA 449 Thr Ala Gly Leu Ser Glu SerSer Asp Pro Leu Glu Leu Val Val Thr 105 110 115 GGA GCC TAC AGC AAA CCCACC CTC TCA GCT CTG CCC AGC CCT GTG GTG 497 Gly Ala Tyr Ser Lys Pro ThrLeu Ser Ala Leu Pro Ser Pro Val Val 120 125 130 135 ACC TCA GGA AGG AATGTG ACC ATC CAG TGT GAC TCA CAG GTG GCA TTT 545 Thr Ser Gly Arg Asn ValThr Ile Gln Cys Asp Ser Gln Val Ala Phe 140 145 150 GAT GGC TTC ATT CTGTGT AAG GAA GGA GAA GAT GAA CAC CCA CAA TGC 593 Asp Gly Phe Ile Leu CysLys Glu Gly Glu Asp Glu His Pro Gln Cys 155 160 165 CTG AAC TCC CAT TCCCAT GCC CGT GGG TCA TCC CGG GCC ATC TTC TCC 641 Leu Asn Ser His Ser HisAla Arg Gly Ser Ser Arg Ala Ile Phe Ser 170 175 180 GTG GGC CCC GTG AGCCCA AGT CGC AGG TGG TCG TAC AGG TGC TAT GGT 689 Val Gly Pro Val Ser ProSer Arg Arg Trp Ser Tyr Arg Cys Tyr Gly 185 190 195 TAT GAC TCG CGC GCTCCC TAT GTG TGG TCT CTA CCC AGT GAT CTC CTG 737 Tyr Asp Ser Arg Ala ProTyr Val Trp Ser Leu Pro Ser Asp Leu Leu 200 205 210 215 GGG CTC CTG GTCCCA GGT GTT TCT AAG AAG CCA TCA CTC TCA GTG CAG 785 Gly Leu Leu Val ProGly Val Ser Lys Lys Pro Ser Leu Ser Val Gln 220 225 230 CCG GGT CCT GTCGTG GCC CCT GGG GAG AAG CTG ACC TTC CAG TGT GGC 833 Pro Gly Pro Val ValAla Pro Gly Glu Lys Leu Thr Phe Gln Cys Gly 235 240 245 TCT GAT GCC GGCTAC GAC AGA TTT GTT CTG TAC AAG GAG TGG GGA CGT 881 Ser Asp Ala Gly TyrAsp Arg Phe Val Leu Tyr Lys Glu Trp Gly Arg 250 255 260 GAC TTC CTC CAGCGC CCT GGC CGG CAG CCC CAG GCT GGG CTC TCC CAG 929 Asp Phe Leu Gln ArgPro Gly Arg Gln Pro Gln Ala Gly Leu Ser Gln 265 270 275 GCC AAC TTC ACCCTG GGC CCT GTG AGC CGC TCC TAC GGG GGC CAG TAC 977 Ala Asn Phe Thr LeuGly Pro Val Ser Arg Ser Tyr Gly Gly Gln Tyr 280 285 290 295 ACA TGC TCCGGT GCA TAC AAC CTC TCC TCC GAG TGG TCG GCC CCC AGC 1025 Thr Cys Ser GlyAla Tyr Asn Leu Ser Ser Glu Trp Ser Ala Pro Ser 300 305 310 GAC CCC CTGGAC ATC CTG ATC ACA GGA CAG ATC CGT GCC AGA CCC TTC 1073 Asp Pro Leu AspIle Leu Ile Thr Gly Gln Ile Arg Ala Arg Pro Phe 315 320 325 CTC TCC GTGCGG CCG GGC CCC ACA GTG GCC TCA GGA GAG AAC GTG ACC 1121 Leu Ser Val ArgPro Gly Pro Thr Val Ala Ser Gly Glu Asn Val Thr 330 335 340 CTG CTG TGTCAG TCA CAG GGA GGG ATG CAC ACT TTC CTT TTG ACC AAG 1169 Leu Leu Cys GlnSer Gln Gly Gly Met His Thr Phe Leu Leu Thr Lys 345 350 355 GAG GGG GCAGCT GAT TCC CCG CTG CGT CTA AAA TCA AAG CGC CAA TCT 1217 Glu Gly Ala AlaAsp Ser Pro Leu Arg Leu Lys Ser Lys Arg Gln Ser 360 365 370 375 CAT AAGTAC CAG GCT GAA TTC CCC ATG AGT CCT GTG ACC TCG GCC CAC 1265 His Lys TyrGln Ala Glu Phe Pro Met Ser Pro Val Thr Ser Ala His 380 385 390 GCG GGGACC TAC AGG TGC TAC GGC TCA CTC AGC TCC AAC CCC TAC CTG 1313 Ala Gly ThrTyr Arg Cys Tyr Gly Ser Leu Ser Ser Asn Pro Tyr Leu 395 400 405 CTG ACTCAC CCC AGT GAC CCC CTG GAG CTC GTG GTC TCA GGA GCA GCT 1361 Leu Thr HisPro Ser Asp Pro Leu Glu Leu Val Val Ser Gly Ala Ala 410 415 420 GAG ACCCTC AGC CCA CCA CAA AAC AAG TCC GAC TCC AAG GCT GGT GAG 1409 Glu Thr LeuSer Pro Pro Gln Asn Lys Ser Asp Ser Lys Ala Gly Glu 425 430 435 TGAGGAGATGCTT GCCGTGATGA CGCTGGGCAC AGAGGGTCAG GTCCTGTCAA 1462 * 440GAGGAGCTGG GTGTCCTGGG TGGACATTTG AAGAATTATA TTCATTCCAA CTTGAAGAAT 1522TATTCAACAC CTTTAACAAT GTATATGTGA AGTACTTTAT TCTTTCATAT TTTAAAAATA 1582AAAGATAATT ATCCATGAGA AAA 1605 439 amino acids amino acid linear protein8 Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Asp 1 5 1015 Pro Arg Thr His Val Gln Ala Gly Pro Leu Pro Lys Pro Thr Leu Trp 20 2530 Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu Arg 35 4045 Cys Gln Gly Ser Leu Glu Thr Gln Glu Tyr His Leu Tyr Arg Glu Lys 50 5560 Lys Thr Ala Leu Trp Ile Thr Arg Ile Pro Gln Glu Leu Val Lys Lys 65 7075 80 Gly Gln Phe Pro Ile Leu Ser Ile Thr Trp Glu His Ala Gly Arg Tyr 8590 95 Cys Cys Ile Tyr Gly Ser His Thr Ala Gly Leu Ser Glu Ser Ser Asp100 105 110 Pro Leu Glu Leu Val Val Thr Gly Ala Tyr Ser Lys Pro Thr LeuSer 115 120 125 Ala Leu Pro Ser Pro Val Val Thr Ser Gly Arg Asn Val ThrIle Gln 130 135 140 Cys Asp Ser Gln Val Ala Phe Asp Gly Phe Ile Leu CysLys Glu Gly 145 150 155 160 Glu Asp Glu His Pro Gln Cys Leu Asn Ser HisSer His Ala Arg Gly 165 170 175 Ser Ser Arg Ala Ile Phe Ser Val Gly ProVal Ser Pro Ser Arg Arg 180 185 190 Trp Ser Tyr Arg Cys Tyr Gly Tyr AspSer Arg Ala Pro Tyr Val Trp 195 200 205 Ser Leu Pro Ser Asp Leu Leu GlyLeu Leu Val Pro Gly Val Ser Lys 210 215 220 Lys Pro Ser Leu Ser Val GlnPro Gly Pro Val Val Ala Pro Gly Glu 225 230 235 240 Lys Leu Thr Phe GlnCys Gly Ser Asp Ala Gly Tyr Asp Arg Phe Val 245 250 255 Leu Tyr Lys GluTrp Gly Arg Asp Phe Leu Gln Arg Pro Gly Arg Gln 260 265 270 Pro Gln AlaGly Leu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser 275 280 285 Arg SerTyr Gly Gly Gln Tyr Thr Cys Ser Gly Ala Tyr Asn Leu Ser 290 295 300 SerGlu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly 305 310 315320 Gln Ile Arg Ala Arg Pro Phe Leu Ser Val Arg Pro Gly Pro Thr Val 325330 335 Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Gln Gly Gly Met340 345 350 His Thr Phe Leu Leu Thr Lys Glu Gly Ala Ala Asp Ser Pro LeuArg 355 360 365 Leu Lys Ser Lys Arg Gln Ser His Lys Tyr Gln Ala Glu PhePro Met 370 375 380 Ser Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg CysTyr Gly Ser 385 390 395 400 Leu Ser Ser Asn Pro Tyr Leu Leu Thr His ProSer Asp Pro Leu Glu 405 410 415 Leu Val Val Ser Gly Ala Ala Glu Thr LeuSer Pro Pro Gln Asn Lys 420 425 430 Ser Asp Ser Lys Ala Gly Glu 435 2221base pairs nucleic acid single linear cDNA pbm8 CDS 184..1977 9GCTCACTGCC ACACGCAGCT CAGCCTGGGC GGCACAGCCA GATGCGAGAT GCGTCTCTGC 60TGATCTGAGT CTGCCTGCAG CATGGACCTG GGTCTTCCCT GAAGCATCTC CAGGGCTGGA 120GGGACGACTG CCATGCACCG AGGGCTCATC CATCCGCAGA GCAGGGCAGT GGGAGGAGAC 180GCC ATG ACC CCC ATC GTC ACA GTC CTG ATC TGT CTC GGG CTG AGT CTG 228 MetThr Pro Ile Val Thr Val Leu Ile Cys Leu Gly Leu Ser Leu 1 5 10 15 GGCCCC AGG ACC CAC GTG CAG ACA GGG ACC ATC CCC AAG CCC ACC CTG 276 Gly ProArg Thr His Val Gln Thr Gly Thr Ile Pro Lys Pro Thr Leu 20 25 30 TGG GCTGAG CCA GAC TCT GTG ATC ACC CAG GGG AGT CCC GTC ACC CTC 324 Trp Ala GluPro Asp Ser Val Ile Thr Gln Gly Ser Pro Val Thr Leu 35 40 45 AGT TGT CAGGGG AGC CTT GAA GCC CAG GAG TAC CGT CTA TAT AGG GAG 372 Ser Cys Gln GlySer Leu Glu Ala Gln Glu Tyr Arg Leu Tyr Arg Glu 50 55 60 AAA AAA TCA GCATCT TGG ATT ACA CGG ATA CGA CCA GAG CTT GTG AAG 420 Lys Lys Ser Ala SerTrp Ile Thr Arg Ile Arg Pro Glu Leu Val Lys 65 70 75 AAC GGC CAG TTC CACATC CCA TCC ATC ACC TGG GAA CAC ACA GGG CGA 468 Asn Gly Gln Phe His IlePro Ser Ile Thr Trp Glu His Thr Gly Arg 80 85 90 95 TAT GGC TGT CAG TATTAC AGC CGC GCT CGG TGG TCT GAG CTC AGT GAC 516 Tyr Gly Cys Gln Tyr TyrSer Arg Ala Arg Trp Ser Glu Leu Ser Asp 100 105 110 CCC CTG GTG CTG GTGATG ACA GGA GCC TAC CCA AAA CCC ACC CTC TCA 564 Pro Leu Val Leu Val MetThr Gly Ala Tyr Pro Lys Pro Thr Leu Ser 115 120 125 GCC CAG CCC AGC CCTGTG GTG ACC TCA GGA GGA AGG GTG ACC CTC CAG 612 Ala Gln Pro Ser Pro ValVal Thr Ser Gly Gly Arg Val Thr Leu Gln 130 135 140 TGT GAG TCA CAG GTGGCA TTT GGC GGC TTC ATT CTG TGT AAG GAA GGA 660 Cys Glu Ser Gln Val AlaPhe Gly Gly Phe Ile Leu Cys Lys Glu Gly 145 150 155 GAA GAT GAA CAC CCACAA TGC CTG AAC TCC CAG CCC CAT GCC CGT GGG 708 Glu Asp Glu His Pro GlnCys Leu Asn Ser Gln Pro His Ala Arg Gly 160 165 170 175 TCG TCC CGC GCCATC TTC TCC GTG GGC CCC GTG AGC CCG AAT CGC AGG 756 Ser Ser Arg Ala IlePhe Ser Val Gly Pro Val Ser Pro Asn Arg Arg 180 185 190 TGG TCG CAC AGGTGC TAT GGT TAT GAC TTG AAC TCT CCC TAT GTG TGG 804 Trp Ser His Arg CysTyr Gly Tyr Asp Leu Asn Ser Pro Tyr Val Trp 195 200 205 TCT TCA CCC AGTGAT CTC CTG GAG CTC CTG GTC CCA GGT GTT TCT AAG 852 Ser Ser Pro Ser AspLeu Leu Glu Leu Leu Val Pro Gly Val Ser Lys 210 215 220 AAG CCA TCA CTCTCA GTG CAG CCG GGT CCT GTC GTG GCC CCT GGG GAA 900 Lys Pro Ser Leu SerVal Gln Pro Gly Pro Val Val Ala Pro Gly Glu 225 230 235 AGC CTG ACC CTCCAG TGT GTC TCT GAT GTC GGC TAT GAC AGA TTT GTT 948 Ser Leu Thr Leu GlnCys Val Ser Asp Val Gly Tyr Asp Arg Phe Val 240 245 250 255 CTG TAC AAGGAG GGG GAA CGT GAC CTT CGC CAG CTC CCT GGC CGG CAG 996 Leu Tyr Lys GluGly Glu Arg Asp Leu Arg Gln Leu Pro Gly Arg Gln 260 265 270 CCC CAG GCTGGG CTC TCC CAG GCC AAC TTC ACC CTG GGC CCT GTG AGC 1044 Pro Gln Ala GlyLeu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val Ser 275 280 285 CGC TCC TACGGG GGC CAG TAC AGA TGC TAC GGT GCA TAC AAC CTC TCC 1092 Arg Ser Tyr GlyGly Gln Tyr Arg Cys Tyr Gly Ala Tyr Asn Leu Ser 290 295 300 TCC GAG TGGTCG GCC CCC AGC GAC CCC CTG GAC ATC CTG ATC ACA GGA 1140 Ser Glu Trp SerAla Pro Ser Asp Pro Leu Asp Ile Leu Ile Thr Gly 305 310 315 CAG ATC CATGGC ACA CCC TTC ATC TCA GTG CAG CCA GGC CCC ACA GTG 1188 Gln Ile His GlyThr Pro Phe Ile Ser Val Gln Pro Gly Pro Thr Val 320 325 330 335 GCC TCAGGA GAG AAC GTG ACC CTG CTG TGT CAG TCA TGG CGG CAG TTC 1236 Ala Ser GlyGlu Asn Val Thr Leu Leu Cys Gln Ser Trp Arg Gln Phe 340 345 350 CAC ACTTTC CTT CTG ACC AAG GCG GGA GCA GCT GAT GCC CCA CTC CGT 1284 His Thr PheLeu Leu Thr Lys Ala Gly Ala Ala Asp Ala Pro Leu Arg 355 360 365 CTA AGATCA ATA CAC GAA TAT CCT AAG TAC CAG GCT GAA TTC CCC ATG 1332 Leu Arg SerIle His Glu Tyr Pro Lys Tyr Gln Ala Glu Phe Pro Met 370 375 380 AGT CCTGTG ACC TCA GCC CAC GCG GGG ACC TAC AGG TGC TAC GGC TCA 1380 Ser Pro ValThr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser 385 390 395 CTC AACTCC GAC CCC TAC CTG CTG TCT CAC CCC AGT GAG CCC CTG GAG 1428 Leu Asn SerAsp Pro Tyr Leu Leu Ser His Pro Ser Glu Pro Leu Glu 400 405 410 415 CTCGTG GTC TCA GGA CCC TCC ATG GGT TCC AGC CCC CCA CCC ACC GGT 1476 Leu ValVal Ser Gly Pro Ser Met Gly Ser Ser Pro Pro Pro Thr Gly 420 425 430 CCCATC TCC ACA CCT GCA GGC CCT GAG GAC CAG CCC CTC ACC CCC ACT 1524 Pro IleSer Thr Pro Ala Gly Pro Glu Asp Gln Pro Leu Thr Pro Thr 435 440 445 GGGTCG GAT CCC CAA AGT GGT CTG GGA AGG CAC CTG GGG GTT GTG ATC 1572 Gly SerAsp Pro Gln Ser Gly Leu Gly Arg His Leu Gly Val Val Ile 450 455 460 GGCATC TTG GTG GCC GTC GTC CTA CTG CTC CTC CTC CTC CTC CTC CTC 1620 Gly IleLeu Val Ala Val Val Leu Leu Leu Leu Leu Leu Leu Leu Leu 465 470 475 TTCCTC ATC CTC CGA CAT CGA CGT CAG GGC AAA CAC TGG ACA TCG ACC 1668 Phe LeuIle Leu Arg His Arg Arg Gln Gly Lys His Trp Thr Ser Thr 480 485 490 495CAG AGA AAG GCT GAT TTC CAA CAT CCT GCA GGG GCT GTG GGG CCA GAG 1716 GlnArg Lys Ala Asp Phe Gln His Pro Ala Gly Ala Val Gly Pro Glu 500 505 510CCC ACA GAC AGA GGC CTG CAG TGG AGG TCC AGC CCA GCT GCC GAC GCC 1764 ProThr Asp Arg Gly Leu Gln Trp Arg Ser Ser Pro Ala Ala Asp Ala 515 520 525CAG GAA GAA AAC CTC TAT GCT GCC GTG AAG GAC ACA CAG CCT GAA GAT 1812 GlnGlu Glu Asn Leu Tyr Ala Ala Val Lys Asp Thr Gln Pro Glu Asp 530 535 540GGG GTG GAG ATG GAC ACT CGG GCT GCT GCA TCT GAA GCC CCC CAG GAT 1860 GlyVal Glu Met Asp Thr Arg Ala Ala Ala Ser Glu Ala Pro Gln Asp 545 550 555GTG ACC TAC GCC CAG CTG CAC AGC TTG ACC CTC AGA CGG AAG GCA ACT 1908 ValThr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Lys Ala Thr 560 565 570575 GAG CCT CCT CCA TCC CAG GAA AGG GAA CCT CCA GCT GAG CCC AGC ATC 1956Glu Pro Pro Pro Ser Gln Glu Arg Glu Pro Pro Ala Glu Pro Ser Ile 580 585590 TAC GCC ACC CTG GCC ATC CAC TAG CCCGGAGGGT ACGCAGACTC CACACTCAGT2010 Tyr Ala Thr Leu Ala Ile His 595 AGAAGGAGAC TCAGGACTGC TGAAGGCACGGGAGCTGCCC CCAGTGGACA CCAATGAACC 2070 CCAGTCAGCC TGGACCCCTA ACAAAGACCATGAGGAGATG CTGGGAACTT TGGGACTCAC 2130 TTGATTCTGC AGTCGAAATA ACTAATATCCCTACATTTTT TAATTAAAGC AACAGACTTC 2190 TCAATAAAAG CAGGTCGTCT CGTTCCAATC T2221 598 amino acids amino acid linear protein 10 Met Thr Pro Ile ValThr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr HisVal Gln Thr Gly Thr Ile Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro AspSer Val Ile Thr Gln Gly Ser Pro Val Thr Leu Ser 35 40 45 Cys Gln Gly SerLeu Glu Ala Gln Glu Tyr Arg Leu Tyr Arg Glu Lys 50 55 60 Lys Ser Ala SerTrp Ile Thr Arg Ile Arg Pro Glu Leu Val Lys Asn 65 70 75 80 Gly Gln PheHis Ile Pro Ser Ile Thr Trp Glu His Thr Gly Arg Tyr 85 90 95 Gly Cys GlnTyr Tyr Ser Arg Ala Arg Trp Ser Glu Leu Ser Asp Pro 100 105 110 Leu ValLeu Val Met Thr Gly Ala Tyr Pro Lys Pro Thr Leu Ser Ala 115 120 125 GlnPro Ser Pro Val Val Thr Ser Gly Gly Arg Val Thr Leu Gln Cys 130 135 140Glu Ser Gln Val Ala Phe Gly Gly Phe Ile Leu Cys Lys Glu Gly Glu 145 150155 160 Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro His Ala Arg Gly Ser165 170 175 Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Asn Arg ArgTrp 180 185 190 Ser His Arg Cys Tyr Gly Tyr Asp Leu Asn Ser Pro Tyr ValTrp Ser 195 200 205 Ser Pro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly ValSer Lys Lys 210 215 220 Pro Ser Leu Ser Val Gln Pro Gly Pro Val Val AlaPro Gly Glu Ser 225 230 235 240 Leu Thr Leu Gln Cys Val Ser Asp Val GlyTyr Asp Arg Phe Val Leu 245 250 255 Tyr Lys Glu Gly Glu Arg Asp Leu ArgGln Leu Pro Gly Arg Gln Pro 260 265 270 Gln Ala Gly Leu Ser Gln Ala AsnPhe Thr Leu Gly Pro Val Ser Arg 275 280 285 Ser Tyr Gly Gly Gln Tyr ArgCys Tyr Gly Ala Tyr Asn Leu Ser Ser 290 295 300 Glu Trp Ser Ala Pro SerAsp Pro Leu Asp Ile Leu Ile Thr Gly Gln 305 310 315 320 Ile His Gly ThrPro Phe Ile Ser Val Gln Pro Gly Pro Thr Val Ala 325 330 335 Ser Gly GluAsn Val Thr Leu Leu Cys Gln Ser Trp Arg Gln Phe His 340 345 350 Thr PheLeu Leu Thr Lys Ala Gly Ala Ala Asp Ala Pro Leu Arg Leu 355 360 365 ArgSer Ile His Glu Tyr Pro Lys Tyr Gln Ala Glu Phe Pro Met Ser 370 375 380Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Leu 385 390395 400 Asn Ser Asp Pro Tyr Leu Leu Ser His Pro Ser Glu Pro Leu Glu Leu405 410 415 Val Val Ser Gly Pro Ser Met Gly Ser Ser Pro Pro Pro Thr GlyPro 420 425 430 Ile Ser Thr Pro Ala Gly Pro Glu Asp Gln Pro Leu Thr ProThr Gly 435 440 445 Ser Asp Pro Gln Ser Gly Leu Gly Arg His Leu Gly ValVal Ile Gly 450 455 460 Ile Leu Val Ala Val Val Leu Leu Leu Leu Leu LeuLeu Leu Leu Phe 465 470 475 480 Leu Ile Leu Arg His Arg Arg Gln Gly LysHis Trp Thr Ser Thr Gln 485 490 495 Arg Lys Ala Asp Phe Gln His Pro AlaGly Ala Val Gly Pro Glu Pro 500 505 510 Thr Asp Arg Gly Leu Gln Trp ArgSer Ser Pro Ala Ala Asp Ala Gln 515 520 525 Glu Glu Asn Leu Tyr Ala AlaVal Lys Asp Thr Gln Pro Glu Asp Gly 530 535 540 Val Glu Met Asp Thr ArgAla Ala Ala Ser Glu Ala Pro Gln Asp Val 545 550 555 560 Thr Tyr Ala GlnLeu His Ser Leu Thr Leu Arg Arg Lys Ala Thr Glu 565 570 575 Pro Pro ProSer Gln Glu Arg Glu Pro Pro Ala Glu Pro Ser Ile Tyr 580 585 590 Ala ThrLeu Ala Ile His 595 2446 base pairs nucleic acid single linear cDNApbm36-2 CDS 171..1037 11 CGCAGCTCAA CCTGAGCTAC ACAGCCAGAT GCGAGATGCTTCTCTGCTGA TCTGAGTCTG 60 CCTGCAGCAT GGACCTTGGT CTTCCCTGAA GCATCTCCAGGGCTGGAGGG ACGACTGCCA 120 TGCACCTAGG GCTTATCCAT CCGCAGAGCA GGGCAGTGGGAGGAGACGCT ATG ACC 176 Met Thr 1 CCC ATC CTC ACG GTC CTG ATC TGT CTC GGGCTG AGT CTG GGC CCC CGG 224 Pro Ile Leu Thr Val Leu Ile Cys Leu Gly LeuSer Leu Gly Pro Arg 5 10 15 ACC CAC GTG CAG GCA GGG ACC CTC CCC AAG CCCACA CTC TGG GCT GAG 272 Thr His Val Gln Ala Gly Thr Leu Pro Lys Pro ThrLeu Trp Ala Glu 20 25 30 CCA GGC TCT GTG ATC ACC CAG GGG AGT CCC GTG ACCCTC TGG TGT CAG 320 Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val Thr LeuTrp Cys Gln 35 40 45 50 GGG ATC CTG GAG ACC CAG GAG TAC CGT CTG TAT AGAGAA AAG AAA ACA 368 Gly Ile Leu Glu Thr Gln Glu Tyr Arg Leu Tyr Arg GluLys Lys Thr 55 60 65 GCA CCC TGG ATT ACA CGG ATC CCA CAG GAG ATT GTG AAGAAG GGC CAG 416 Ala Pro Trp Ile Thr Arg Ile Pro Gln Glu Ile Val Lys LysGly Gln 70 75 80 TTC CCC ATC CCG TCC ATC ACC TGG GAA CAC ACC GGG CGG TATCGC TGT 464 Phe Pro Ile Pro Ser Ile Thr Trp Glu His Thr Gly Arg Tyr ArgCys 85 90 95 TTC TAC GGT AGC CAC ACT GCA GGC TGG TCA GAG CCC AGT GAC CCCCTG 512 Phe Tyr Gly Ser His Thr Ala Gly Trp Ser Glu Pro Ser Asp Pro Leu100 105 110 GAG CTG GTG GTG ACA GGA GCC TAC ATC AAA CCC ACC CTC TCG GCTCTA 560 Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser Ala Leu115 120 125 130 CCC AGC CCT GTG GTG ACC TCA GGA GGG AAC GTG ACC CTC CATTGT GTC 608 Pro Ser Pro Val Val Thr Ser Gly Gly Asn Val Thr Leu His CysVal 135 140 145 TCA CAG GTG GCA TTT GGC AGC TTC ATT CTG TGT AAG GAA GGAGAA GAT 656 Ser Gln Val Ala Phe Gly Ser Phe Ile Leu Cys Lys Glu Gly GluAsp 150 155 160 GAA CAC CCA CAA TGC CTG AAC TCA CAG CCC CGT ACC CAT GGGTGG TCC 704 Glu His Pro Gln Cys Leu Asn Ser Gln Pro Arg Thr His Gly TrpSer 165 170 175 CGG GCC ATC TTC TCT GTG GGC CCC GTG AGC CCG AGT CGC AGGTGG TCG 752 Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg TrpSer 180 185 190 TAC AGG TGC TAT GCT TAT GAC TCG AAC TCT CCC CAT GTG TGGTCT CTA 800 Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro His Val Trp SerLeu 195 200 205 210 CCC AGT GAT CTC CTG GAG CTC CTG GTC CCA GGA GCA GCTGAG ACC CTC 848 Pro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly Ala Ala GluThr Leu 215 220 225 AGC CCA CCA CAA AAC AAG TCC GAT TCC AAG GCT GGA GCAGCT AAC ACC 896 Ser Pro Pro Gln Asn Lys Ser Asp Ser Lys Ala Gly Ala AlaAsn Thr 230 235 240 CTC AGC CCA TCA CAA AAC AAG ACT GCC TCA CAC CCC CAGGAT TAC ACA 944 Leu Ser Pro Ser Gln Asn Lys Thr Ala Ser His Pro Gln AspTyr Thr 245 250 255 GTG GAG AAT CTC ATC CGC ATG GGC ATA GCT GGC TTG GTCCTG GTG GTC 992 Val Glu Asn Leu Ile Arg Met Gly Ile Ala Gly Leu Val LeuVal Val 260 265 270 CTC GGG ATT CTG CTA TTT GAG GCT CAG CAC AGC CAG AGAAGC CTC TGA 1040 Leu Gly Ile Leu Leu Phe Glu Ala Gln His Ser Gln Arg SerLeu 275 280 285 GATGCAGCCG GGAGGTGAAC AGCAGAGAGA AGAATGTACC CTTCAGAGTGGTGGAGCCTT 1100 GGGAACAGAT CTGATGATGC CAGGAGGTTC CGGGAGACAA TTTAGGGCTGATGCTATCTG 1160 GACTGTCTGC CAATCATTTT TAGAGGGAGG AATCAGTGTT GGATTGCAGAGACATTTTCT 1220 GGAGTGATCC ATGAAGGACC ATTAACATGT GATACCTTTC CTCTCTATTAATGTTGACTT 1280 CCCTTGGTTG GATCCTCTTC TTTCCCCACC CCCAGACAGA CATGAGGCTACATCCCACAT 1340 GGCAGCGTTG GGTCCACACC TCTGCACATC TGTGTGCTCT GGTCCATGGTGTGTAACACA 1400 GTCTTCTTTA TTACTCATTG CCATACTCCC TGGTGTGCTT TACTGAGCCTCCATCTCTTC 1460 AATTCAGAGT TCCAAACGTG CTTCAGTAAC TAAATCAATG GGAGAGTATCGGATTTCAAC 1520 CAGGAAAAGA TAAATCCACC CTGATGCCCT GACACCCTCT CTGAACCCTACGAGCCCTTC 1580 CCTCCTTCTC ACATGCTACC TGTGCAGCTT CTCCTTAGAT CATTGTGTAACCATCACTGC 1640 CATCCTGTTC CACACATGGT CATCACCCTA CACCCATTCA GCAGCCACTCCCCATTCCCT 1700 CTTCCCTCCA GCACCTGCTA ACCACAAATG TGCTTTCTGT CTCTACGGATTTGCCTATTC 1760 TGTCTGAAAA CATTTCAATC TCCTTTGACC TGTGAGCTCC TCACTTCGAGACTTCCTGCC 1820 TTTCCAGGCA GAACCAAAGT ACACCACGTC AAAAGCAATG ATAGGCATTTGCAGTGTGTT 1880 GGTGATCCAC GAAAGGAAAA TCACGGAAGC AGGATAGAAA TCCAGCTGCAGACAAGACCT 1940 CAGGTCGATG AATCTTGACA AGCAGTTGAG CTGTTTTTTT CTACTCACCTAGGACAGTCA 2000 GGCAGAAGTA TGCAAAATGA CTGGGGCTGA TTCTTTTCTG AATTGTCGCAAACAGCAAGA 2060 GGACTTGAGT CCTAGCATTA AAGAGTTCAA CATGTCTAGG TCCAAGACCACTGTTGTGTT 2120 TGAAGGATGT AAAACCCTGC TGCATAGGAT GGAATATTTG GAGGGAGGATCCTGAAAAAC 2180 ATGAGGGATC AAATAGTCCT CAACTTTCTA GGACAAAGGG AGCAGCTATTTGCCATCTAC 2240 CCTCCAGAAT AAAGAAATCT TATCATTCAC CATCTACCCT CTAGAATAAAGAAATCTTAT 2300 CATTCGCCAT CTACCCTGTA GAATAAAGAA ATCTTATCAT TCACCGTCTACCCTCTAGAG 2360 TAAACAAATC TTATCATTCA CCATCTACCC TCTAGAATAA AGAAATCTTATCATTCGCCA 2420 TCTACCCTCT AGAATAAAGA AATCTT 2446 289 amino acids aminoacid linear protein 12 Met Thr Pro Ile Leu Thr Val Leu Ile Cys Leu GlyLeu Ser Leu Gly 1 5 10 15 Pro Arg Thr His Val Gln Ala Gly Thr Leu ProLys Pro Thr Leu Trp 20 25 30 Ala Glu Pro Gly Ser Val Ile Thr Gln Gly SerPro Val Thr Leu Trp 35 40 45 Cys Gln Gly Ile Leu Glu Thr Gln Glu Tyr ArgLeu Tyr Arg Glu Lys 50 55 60 Lys Thr Ala Pro Trp Ile Thr Arg Ile Pro GlnGlu Ile Val Lys Lys 65 70 75 80 Gly Gln Phe Pro Ile Pro Ser Ile Thr TrpGlu His Thr Gly Arg Tyr 85 90 95 Arg Cys Phe Tyr Gly Ser His Thr Ala GlyTrp Ser Glu Pro Ser Asp 100 105 110 Pro Leu Glu Leu Val Val Thr Gly AlaTyr Ile Lys Pro Thr Leu Ser 115 120 125 Ala Leu Pro Ser Pro Val Val ThrSer Gly Gly Asn Val Thr Leu His 130 135 140 Cys Val Ser Gln Val Ala PheGly Ser Phe Ile Leu Cys Lys Glu Gly 145 150 155 160 Glu Asp Glu His ProGln Cys Leu Asn Ser Gln Pro Arg Thr His Gly 165 170 175 Trp Ser Arg AlaIle Phe Ser Val Gly Pro Val Ser Pro Ser Arg Arg 180 185 190 Trp Ser TyrArg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro His Val Trp 195 200 205 Ser LeuPro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly Ala Ala Glu 210 215 220 ThrLeu Ser Pro Pro Gln Asn Lys Ser Asp Ser Lys Ala Gly Ala Ala 225 230 235240 Asn Thr Leu Ser Pro Ser Gln Asn Lys Thr Ala Ser His Pro Gln Asp 245250 255 Tyr Thr Val Glu Asn Leu Ile Arg Met Gly Ile Ala Gly Leu Val Leu260 265 270 Val Val Leu Gly Ile Leu Leu Phe Glu Ala Gln His Ser Gln ArgSer 275 280 285 Leu 1910 base pairs nucleic acid single linear cDNApbm36-4 CDS 183..1649 13 CTCACTGCCA CACGCAGCTC AACCTGAGCT ACACAGCCAGATGCGAGATG CTTCTCTGCT 60 GATCTGAGTC TGCCTGCAGC ATGGACCTTG GTCTTCCCTGAAGCATCTCC AGGGCTGGAG 120 GGACGACTGC CATGCACCGA GGGCTCATCC ATCCGCAGAGCAGGGCAGTG GGAGGAGACG 180 CT ATG ACC CCC ATC GTC ACA GTC CTG ATC TGT CTCAGG CTG AGT CTG 227 Met Thr Pro Ile Val Thr Val Leu Ile Cys Leu Arg LeuSer Leu 1 5 10 15 GGC CCC CGG ACC CAC GTG CAG GCA GGG ACC CTC CCC AAGCCC ACA CTC 275 Gly Pro Arg Thr His Val Gln Ala Gly Thr Leu Pro Lys ProThr Leu 20 25 30 TGG GCT GAG CCA GGC TCT GTG ATC ACC CAG GGG AGT CCC GTGACC CTC 323 Trp Ala Glu Pro Gly Ser Val Ile Thr Gln Gly Ser Pro Val ThrLeu 35 40 45 TGG TGT CAG GGG ATC CTG GAG ACC CAG GAG TAC CGT CTG TAT AGAGAA 371 Trp Cys Gln Gly Ile Leu Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu50 55 60 AAG AAA ACA GCA CCC TGG ATT ACA CGG ATC CCA CAG GAG ATT GTG AAG419 Lys Lys Thr Ala Pro Trp Ile Thr Arg Ile Pro Gln Glu Ile Val Lys 6570 75 AAG GGC CAG TTC CCC ATC CCA TCC ATC ACC TGG GAA CAC ACA GGG CGG467 Lys Gly Gln Phe Pro Ile Pro Ser Ile Thr Trp Glu His Thr Gly Arg 8085 90 95 TAT CGC TGT TTC TAC GGT AGC CAC ACT GCA GGC TGG TCA GAG CCC AGT515 Tyr Arg Cys Phe Tyr Gly Ser His Thr Ala Gly Trp Ser Glu Pro Ser 100105 110 GAC CCC CTG GAG CTG GTG GTG ACA GGA GCC TAC ATC AAA CCC ACC CTC563 Asp Pro Leu Glu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu 115120 125 TCA GCT CTA CCC AGC CCT GTG GTG ACC TCA GGA GGG AAC GTG ACC CTC611 Ser Ala Leu Pro Ser Pro Val Val Thr Ser Gly Gly Asn Val Thr Leu 130135 140 CAT TGT GTC TCA CAG GTG GCA TTT GGC AGC TTC ATT CTG TGT AAG GAA659 His Cys Val Ser Gln Val Ala Phe Gly Ser Phe Ile Leu Cys Lys Glu 145150 155 GGA GAA GAT GAA CAC CCA CAA TGC CTG AAC TCA CAG CCC CGT ACC CAT707 Gly Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro Arg Thr His 160165 170 175 GGG TGG TCC CGG GCC ATC TTC TCT GTG GGC CCC GTG AGC CCG AGTCGC 755 Gly Trp Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg180 185 190 AGG TGG TCG TAC AGG TGC TAT GCT TAT GAC TCG AAC TCT CCC CATGTG 803 Arg Trp Ser Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro His Val195 200 205 TGG TCT CTA CCC AGT GAT CTC CTG GAG CTC CTG GTC CTA GGT GTTTCT 851 Trp Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu Gly Val Ser210 215 220 AAG AAG CCA TCA CTC TCA GTG CAG CCA GGT CCT ATA GTG GCC CCTGGG 899 Lys Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Ile Val Ala Pro Gly225 230 235 GAG AGC CTG ACC CTC CAG TGT GTT TCT GAT GTC AGC TAC GAC AGATTT 947 Glu Ser Leu Thr Leu Gln Cys Val Ser Asp Val Ser Tyr Asp Arg Phe240 245 250 255 GTT CTG TAT AAG GAG GGA GAA CGT GAC TTC CTC CAG CTC CCTGGC CCA 995 Val Leu Tyr Lys Glu Gly Glu Arg Asp Phe Leu Gln Leu Pro GlyPro 260 265 270 CAG CCC CAG GCT GGG CTC TCC CAG GCC AAC TTC ACC CTG GGCCCT GTG 1043 Gln Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly ProVal 275 280 285 AGC CGC TCC TAC GGG GGC CAG TAC AGA TGC TCC GGT GCA TACAAC CTC 1091 Ser Arg Ser Tyr Gly Gly Gln Tyr Arg Cys Ser Gly Ala Tyr AsnLeu 290 295 300 TCC TCC GAG TGG TCG GCC CCC AGC GAC CCC CTG GAC ATC CTGATC GCA 1139 Ser Ser Glu Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu IleAla 305 310 315 GGA CAG TTC CGT GGC AGA CCC TTC ATC TCG GTG CAT CCG GGCCCC ACG 1187 Gly Gln Phe Arg Gly Arg Pro Phe Ile Ser Val His Pro Gly ProThr 320 325 330 335 GTG GCC TCA GGA GAG AAC GTG ACC CTG CTG TGT CAG TCATGG GGG CCG 1235 Val Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser TrpGly Pro 340 345 350 TTC CAC ACT TTC CTT CTG ACC AAG GCG GGA GCA GCT GATGCC CCC CTC 1283 Phe His Thr Phe Leu Leu Thr Lys Ala Gly Ala Ala Asp AlaPro Leu 355 360 365 CGT CTC AGA TCA ATA CAC GAA TAT CCT AAG TAC CAG GCTGAA TTC CCT 1331 Arg Leu Arg Ser Ile His Glu Tyr Pro Lys Tyr Gln Ala GluPhe Pro 370 375 380 ATG AGT CCT GTG ACC TCA GCC CAC TCG GGG ACC TAC AGGTGC TAC GGC 1379 Met Ser Pro Val Thr Ser Ala His Ser Gly Thr Tyr Arg CysTyr Gly 385 390 395 TCA CTC AGC TCC AAC CCC TAC CTG CTG TCT CAC CCC AGTGAC TCC CTG 1427 Ser Leu Ser Ser Asn Pro Tyr Leu Leu Ser His Pro Ser AspSer Leu 400 405 410 415 GAG CTC ATG GTC TCA GGA GCA GCT GAG ACC CTC AGCCCA CCA CAA AAC 1475 Glu Leu Met Val Ser Gly Ala Ala Glu Thr Leu Ser ProPro Gln Asn 420 425 430 AAG TCC GAT TCC AAG GCT GGA GCA GCT AAC ACC CTCAGC CCA TCA CAA 1523 Lys Ser Asp Ser Lys Ala Gly Ala Ala Asn Thr Leu SerPro Ser Gln 435 440 445 AAC AAG ACT GCC TCA CAC CCC CAG GAT TAC ACA GTGGAG AAT CTC ATC 1571 Asn Lys Thr Ala Ser His Pro Gln Asp Tyr Thr Val GluAsn Leu Ile 450 455 460 CGC ATG GGC ATA GCT GGC TTG GTC CTG GTG GTC CTCGGG ATT CTG CTA 1619 Arg Met Gly Ile Ala Gly Leu Val Leu Val Val Leu GlyIle Leu Leu 465 470 475 TTT GAG GCT CAG CAC AGC CAG AGA AGC CTC TGAGATGCAGCCG GGAGGTGAAC 1672 Phe Glu Ala Gln His Ser Gln Arg Ser Leu 480485 AGCAGAGAGA AGAATGTACC CTTCAGAGTG GTGGAGCCTT GGGAACAGAT CTGATGATGC1732 CAGGAGGTTC CGGGAGACAA TTTAGGGCTG ATGTTATCTG GACTGTCTGC CAATCATTTT1792 TAGAGGGAGG AATCAGTGTT GGATTGCAGA GACATTTTCT GGAGTGATCC ATGAAGGACC1852 ATTAACATGT GATACCTTTC CTCTCTATTA ATGTTGACTT CCCTTGGTTG GATCCTCT1910 489 amino acids amino acid linear protein 14 Met Thr Pro Ile ValThr Val Leu Ile Cys Leu Arg Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr HisVal Gln Ala Gly Thr Leu Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro GlySer Val Ile Thr Gln Gly Ser Pro Val Thr Leu Trp 35 40 45 Cys Gln Gly IleLeu Glu Thr Gln Glu Tyr Arg Leu Tyr Arg Glu Lys 50 55 60 Lys Thr Ala ProTrp Ile Thr Arg Ile Pro Gln Glu Ile Val Lys Lys 65 70 75 80 Gly Gln PhePro Ile Pro Ser Ile Thr Trp Glu His Thr Gly Arg Tyr 85 90 95 Arg Cys PheTyr Gly Ser His Thr Ala Gly Trp Ser Glu Pro Ser Asp 100 105 110 Pro LeuGlu Leu Val Val Thr Gly Ala Tyr Ile Lys Pro Thr Leu Ser 115 120 125 AlaLeu Pro Ser Pro Val Val Thr Ser Gly Gly Asn Val Thr Leu His 130 135 140Cys Val Ser Gln Val Ala Phe Gly Ser Phe Ile Leu Cys Lys Glu Gly 145 150155 160 Glu Asp Glu His Pro Gln Cys Leu Asn Ser Gln Pro Arg Thr His Gly165 170 175 Trp Ser Arg Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser ArgArg 180 185 190 Trp Ser Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro HisVal Trp 195 200 205 Ser Leu Pro Ser Asp Leu Leu Glu Leu Leu Val Leu GlyVal Ser Lys 210 215 220 Lys Pro Ser Leu Ser Val Gln Pro Gly Pro Ile ValAla Pro Gly Glu 225 230 235 240 Ser Leu Thr Leu Gln Cys Val Ser Asp ValSer Tyr Asp Arg Phe Val 245 250 255 Leu Tyr Lys Glu Gly Glu Arg Asp PheLeu Gln Leu Pro Gly Pro Gln 260 265 270 Pro Gln Ala Gly Leu Ser Gln AlaAsn Phe Thr Leu Gly Pro Val Ser 275 280 285 Arg Ser Tyr Gly Gly Gln TyrArg Cys Ser Gly Ala Tyr Asn Leu Ser 290 295 300 Ser Glu Trp Ser Ala ProSer Asp Pro Leu Asp Ile Leu Ile Ala Gly 305 310 315 320 Gln Phe Arg GlyArg Pro Phe Ile Ser Val His Pro Gly Pro Thr Val 325 330 335 Ala Ser GlyGlu Asn Val Thr Leu Leu Cys Gln Ser Trp Gly Pro Phe 340 345 350 His ThrPhe Leu Leu Thr Lys Ala Gly Ala Ala Asp Ala Pro Leu Arg 355 360 365 LeuArg Ser Ile His Glu Tyr Pro Lys Tyr Gln Ala Glu Phe Pro Met 370 375 380Ser Pro Val Thr Ser Ala His Ser Gly Thr Tyr Arg Cys Tyr Gly Ser 385 390395 400 Leu Ser Ser Asn Pro Tyr Leu Leu Ser His Pro Ser Asp Ser Leu Glu405 410 415 Leu Met Val Ser Gly Ala Ala Glu Thr Leu Ser Pro Pro Gln AsnLys 420 425 430 Ser Asp Ser Lys Ala Gly Ala Ala Asn Thr Leu Ser Pro SerGln Asn 435 440 445 Lys Thr Ala Ser His Pro Gln Asp Tyr Thr Val Glu AsnLeu Ile Arg 450 455 460 Met Gly Ile Ala Gly Leu Val Leu Val Val Leu GlyIle Leu Leu Phe 465 470 475 480 Glu Ala Gln His Ser Gln Arg Ser Leu 4851725 base pairs nucleic acid single linear cDNA pbmhh CDS 40..1488 15CTCATCCATC CGCAGAGCAG GGCAGTGGGA GGAGACGCC ATG ACC CCC ATC CTC 54 MetThr Pro Ile Leu 1 5 ACG GTC CTG ATC TGT CTC GGG CTG AGT CTG GGC CCC AGGACC CAC GTG 102 Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly Pro Arg ThrHis Val 10 15 20 CAG GCA GGG CAC CTC CCC AAG CCC ACC CTC TGG GCT GAG CCAGGC TCT 150 Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp Ala Glu Pro GlySer 25 30 35 GTG ATC ATC CAG GGA AGT CCT GTG ACC CTC AGG TGT CAG GGG AGCCTT 198 Val Ile Ile Gln Gly Ser Pro Val Thr Leu Arg Cys Gln Gly Ser Leu40 45 50 CAG GCT GAG GAG TAC CAT CTA TAT AGG GAA AAC AAA TCA GCA TCC TGG246 Gln Ala Glu Glu Tyr His Leu Tyr Arg Glu Asn Lys Ser Ala Ser Trp 5560 65 GTT AGA CGG ATA CAA GAG CCT GGG AAG AAT GGC CAG TTC CCC ATC CCA294 Val Arg Arg Ile Gln Glu Pro Gly Lys Asn Gly Gln Phe Pro Ile Pro 7075 80 85 TCC ATC ACC TGG GAA CAC GCA GGG CGG TAT CAC TGT CAG TAC TAC AGC342 Ser Ile Thr Trp Glu His Ala Gly Arg Tyr His Cys Gln Tyr Tyr Ser 9095 100 CAC AAT CAC TCA TCA GAG TAC AGT GAC CCC CTG GAG CTG GTG GTG ACA390 His Asn His Ser Ser Glu Tyr Ser Asp Pro Leu Glu Leu Val Val Thr 105110 115 GGA GCC TAC AGC AAA CCC ACC CTC TCA GCT CTG CCC AGC CCT GTG GTG438 Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro Val Val 120125 130 ACC TTA GGA GGG AAC GTG ACC CTC CAG TGT GTC TCA CAG GTG GCA TTT486 Thr Leu Gly Gly Asn Val Thr Leu Gln Cys Val Ser Gln Val Ala Phe 135140 145 GAC GGC TTC ATT CTG TGT AAG GAA GGA GAA GAT GAA CAC CCA CAA CGC534 Asp Gly Phe Ile Leu Cys Lys Glu Gly Glu Asp Glu His Pro Gln Arg 150155 160 165 CTG AAC TCC CAT TCC CAT GCC CGT GGG TGG TCC TGG GCC ATC TTCTCC 582 Leu Asn Ser His Ser His Ala Arg Gly Trp Ser Trp Ala Ile Phe Ser170 175 180 GTG GGC CCC GTG AGC CCG AGT CGC AGG TGG TCG TAC AGG TGC TATGCT 630 Val Gly Pro Val Ser Pro Ser Arg Arg Trp Ser Tyr Arg Cys Tyr Ala185 190 195 TAT GAC TCG AAC TCT CCC TAT GTG TGG TCT CTA CCC AGT GAT CTCCTG 678 Tyr Asp Ser Asn Ser Pro Tyr Val Trp Ser Leu Pro Ser Asp Leu Leu200 205 210 GAG CTC CTG GTC CCA GGT GTT TCT AAG AAG CCA TCA CTC TCA GTGCAG 726 Glu Leu Leu Val Pro Gly Val Ser Lys Lys Pro Ser Leu Ser Val Gln215 220 225 CCA GGT CCT ATG GTG GCC CCC GGG GAG AGC CTG ACC CTC CAG TGTGTC 774 Pro Gly Pro Met Val Ala Pro Gly Glu Ser Leu Thr Leu Gln Cys Val230 235 240 245 TCT GAT GTC GGC TAC GAC AGA TTT GTT CTG TAT AAG GAG GGAGAA CGT 822 Ser Asp Val Gly Tyr Asp Arg Phe Val Leu Tyr Lys Glu Gly GluArg 250 255 260 GAC TTC CTC CAG CGC CCT GGT TGG CAG CCC CAG GCT GGG CTCTCC CAG 870 Asp Phe Leu Gln Arg Pro Gly Trp Gln Pro Gln Ala Gly Leu SerGln 265 270 275 GCC AAC TTC ACC CTG GGC CCT GTG AGC CCC TCC CAC GGG GGCCAG TAC 918 Ala Asn Phe Thr Leu Gly Pro Val Ser Pro Ser His Gly Gly GlnTyr 280 285 290 AGA TGC TAC AGT GCA CAC AAC CTC TCC TCC GAG TGG TCG GCCCCC AGT 966 Arg Cys Tyr Ser Ala His Asn Leu Ser Ser Glu Trp Ser Ala ProSer 295 300 305 GAC CCC CTG GAC ATC CTG ATC ACA GGA CAG TTC TAT GAC AGACCC TCT 1014 Asp Pro Leu Asp Ile Leu Ile Thr Gly Gln Phe Tyr Asp Arg ProSer 310 315 320 325 CTC TCG GTG CAG CCG GTC CCC ACA GTA GCC CCA GGA AAGAAC GTG ACC 1062 Leu Ser Val Gln Pro Val Pro Thr Val Ala Pro Gly Lys AsnVal Thr 330 335 340 CTG CTG TGT CAG TCA CGG GGG CAG TTC CAC ACT TTC CTTCTG ACC AAG 1110 Leu Leu Cys Gln Ser Arg Gly Gln Phe His Thr Phe Leu LeuThr Lys 345 350 355 GAG GGG GCA GGC CAT CCC CCA CTG CAT CTG AGA TCA GAGCAC CAA GCT 1158 Glu Gly Ala Gly His Pro Pro Leu His Leu Arg Ser Glu HisGln Ala 360 365 370 CAG CAG AAC CAG GCT GAA TTC CGC ATG GGT CCT GTG ACCTCA GCC CAC 1206 Gln Gln Asn Gln Ala Glu Phe Arg Met Gly Pro Val Thr SerAla His 375 380 385 GTG GGG ACC TAC AGA TGC TAC AGC TCA CTC AGC TCC AACCCC TAC CTG 1254 Val Gly Thr Tyr Arg Cys Tyr Ser Ser Leu Ser Ser Asn ProTyr Leu 390 395 400 405 CTG TCT CTC CCC AGT GAC CCC CTG GAG CTC GTG GTCTCA GAA GCA GCT 1302 Leu Ser Leu Pro Ser Asp Pro Leu Glu Leu Val Val SerGlu Ala Ala 410 415 420 GAG ACC CTC AGC CCA TCA CAA AAC AAG ACA GAC TCCACG ACT ACA TCC 1350 Glu Thr Leu Ser Pro Ser Gln Asn Lys Thr Asp Ser ThrThr Thr Ser 425 430 435 CTA GGC CAA CAC CCC CAG GAT TAC ACA GTG GAG AATCTC ATC CGC ATG 1398 Leu Gly Gln His Pro Gln Asp Tyr Thr Val Glu Asn LeuIle Arg Met 440 445 450 GGT GTG GCT GGC TTG GTC CTG GTG GTC CTC GGG ATTCTG CTA TTT GAG 1446 Gly Val Ala Gly Leu Val Leu Val Val Leu Gly Ile LeuLeu Phe Glu 455 460 465 GCT CAG CAC AGC CAG AGA AGC CTA CAA GAT GCA GCCGGG AGG TGA 1491 Ala Gln His Ser Gln Arg Ser Leu Gln Asp Ala Ala Gly Arg470 475 480 ACAGCAGAGA GGACAATGCA TCCTTCAGCG TGGTGGAGCC TCAGGGACAGATCTGATGAT 1551 CCCAGGAGGC TCTGGAGGAC AATCTAGGAC CTACATTATC TGGACTGTATGCTGGTCATT 1611 TCTAGAGACA GCAATCAATA TTTGAGTGTA AGGAAACTGT CTGGGGTGATTCCTAGAAGA 1671 TCATTAAACT GTGGTACATT TTTTTGTCTA AAAAGCAGGT CGTCTCGTTCCAAG 1725 483 amino acids amino acid linear protein 16 Met Thr Pro IleLeu Thr Val Leu Ile Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg ThrHis Val Gln Ala Gly His Leu Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu ProGly Ser Val Ile Ile Gln Gly Ser Pro Val Thr Leu Arg 35 40 45 Cys Gln GlySer Leu Gln Ala Glu Glu Tyr His Leu Tyr Arg Glu Asn 50 55 60 Lys Ser AlaSer Trp Val Arg Arg Ile Gln Glu Pro Gly Lys Asn Gly 65 70 75 80 Gln PhePro Ile Pro Ser Ile Thr Trp Glu His Ala Gly Arg Tyr His 85 90 95 Cys GlnTyr Tyr Ser His Asn His Ser Ser Glu Tyr Ser Asp Pro Leu 100 105 110 GluLeu Val Val Thr Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala Leu 115 120 125Pro Ser Pro Val Val Thr Leu Gly Gly Asn Val Thr Leu Gln Cys Val 130 135140 Ser Gln Val Ala Phe Asp Gly Phe Ile Leu Cys Lys Glu Gly Glu Asp 145150 155 160 Glu His Pro Gln Arg Leu Asn Ser His Ser His Ala Arg Gly TrpSer 165 170 175 Trp Ala Ile Phe Ser Val Gly Pro Val Ser Pro Ser Arg ArgTrp Ser 180 185 190 Tyr Arg Cys Tyr Ala Tyr Asp Ser Asn Ser Pro Tyr ValTrp Ser Leu 195 200 205 Pro Ser Asp Leu Leu Glu Leu Leu Val Pro Gly ValSer Lys Lys Pro 210 215 220 Ser Leu Ser Val Gln Pro Gly Pro Met Val AlaPro Gly Glu Ser Leu 225 230 235 240 Thr Leu Gln Cys Val Ser Asp Val GlyTyr Asp Arg Phe Val Leu Tyr 245 250 255 Lys Glu Gly Glu Arg Asp Phe LeuGln Arg Pro Gly Trp Gln Pro Gln 260 265 270 Ala Gly Leu Ser Gln Ala AsnPhe Thr Leu Gly Pro Val Ser Pro Ser 275 280 285 His Gly Gly Gln Tyr ArgCys Tyr Ser Ala His Asn Leu Ser Ser Glu 290 295 300 Trp Ser Ala Pro SerAsp Pro Leu Asp Ile Leu Ile Thr Gly Gln Phe 305 310 315 320 Tyr Asp ArgPro Ser Leu Ser Val Gln Pro Val Pro Thr Val Ala Pro 325 330 335 Gly LysAsn Val Thr Leu Leu Cys Gln Ser Arg Gly Gln Phe His Thr 340 345 350 PheLeu Leu Thr Lys Glu Gly Ala Gly His Pro Pro Leu His Leu Arg 355 360 365Ser Glu His Gln Ala Gln Gln Asn Gln Ala Glu Phe Arg Met Gly Pro 370 375380 Val Thr Ser Ala His Val Gly Thr Tyr Arg Cys Tyr Ser Ser Leu Ser 385390 395 400 Ser Asn Pro Tyr Leu Leu Ser Leu Pro Ser Asp Pro Leu Glu LeuVal 405 410 415 Val Ser Glu Ala Ala Glu Thr Leu Ser Pro Ser Gln Asn LysThr Asp 420 425 430 Ser Thr Thr Thr Ser Leu Gly Gln His Pro Gln Asp TyrThr Val Glu 435 440 445 Asn Leu Ile Arg Met Gly Val Ala Gly Leu Val LeuVal Val Leu Gly 450 455 460 Ile Leu Leu Phe Glu Ala Gln His Ser Gln ArgSer Leu Gln Asp Ala 465 470 475 480 Ala Gly Arg 1625 base pairs nucleicacid single linear cDNA pbm2 CDS 30..1373 17 CACAGCTGGG GCCCCTGGGAGGAGACGCC ATG ATC CCC ACC TTC ACG GCT CTG 53 Met Ile Pro Thr Phe Thr AlaLeu 1 5 CTC TGC CTC GGG CTG AGT CTG GGC CCC AGG ACC CAC ATG CAG GCA GGG101 Leu Cys Leu Gly Leu Ser Leu Gly Pro Arg Thr His Met Gln Ala Gly 1015 20 CCC CTC CCC AAA CCC ACC CTC TGG GCT GAG CCA GGC TCT GTG ATC AGC149 Pro Leu Pro Lys Pro Thr Leu Trp Ala Glu Pro Gly Ser Val Ile Ser 2530 35 40 TGG GGG AAC TCT GTG ACC ATC TGG TGT CAG GGG ACC CTG GAG GCT CGG197 Trp Gly Asn Ser Val Thr Ile Trp Cys Gln Gly Thr Leu Glu Ala Arg 4550 55 GAG TAC CGT CTG GAT AAA GAG GAA AGC CCA GCA CCC TGG GAC AGA CAG245 Glu Tyr Arg Leu Asp Lys Glu Glu Ser Pro Ala Pro Trp Asp Arg Gln 6065 70 AAC CCA CTG GAG CCC AAG AAC AAG GCC AGA TTC TCC ATC CCA TCC ATG293 Asn Pro Leu Glu Pro Lys Asn Lys Ala Arg Phe Ser Ile Pro Ser Met 7580 85 ACA GAG GAC TAT GCA GGG AGA TAC CGC TGT TAC TAT CGC AGC CCT GTA341 Thr Glu Asp Tyr Ala Gly Arg Tyr Arg Cys Tyr Tyr Arg Ser Pro Val 9095 100 GGC TGG TCA CAG CCC AGT GAC CCC CTG GAG CTG GTG ATG ACA GGA GCC389 Gly Trp Ser Gln Pro Ser Asp Pro Leu Glu Leu Val Met Thr Gly Ala 105110 115 120 TAC AGT AAA CCC ACC CTT TCA GCC CTG CCG AGT CCT CTT GTG ACCTCA 437 Tyr Ser Lys Pro Thr Leu Ser Ala Leu Pro Ser Pro Leu Val Thr Ser125 130 135 GGA AAG AGC GTG ACC CTG CTG TGT CAG TCA CGG AGC CCA ATG GACACT 485 Gly Lys Ser Val Thr Leu Leu Cys Gln Ser Arg Ser Pro Met Asp Thr140 145 150 TTT CTT CTG ATC AAG GAG CGG GCA GCC CAT CCC CTA CTG CAT CTGAGA 533 Phe Leu Leu Ile Lys Glu Arg Ala Ala His Pro Leu Leu His Leu Arg155 160 165 TCA GAG CAC GGA GCT CAG CAG CAC CAG GCT GAA TTC CCC ATG AGTCCT 581 Ser Glu His Gly Ala Gln Gln His Gln Ala Glu Phe Pro Met Ser Pro170 175 180 GTG ACC TCA GTG CAC GGG GGG ACC TAC AGG TGC TTC AGC TCA CACGGC 629 Val Thr Ser Val His Gly Gly Thr Tyr Arg Cys Phe Ser Ser His Gly185 190 195 200 TTC TCC CAC TAC CTG CTG TCA CAC CCC AGT GAC CCC CTG GAGCTC ATA 677 Phe Ser His Tyr Leu Leu Ser His Pro Ser Asp Pro Leu Glu LeuIle 205 210 215 GTC TCA GGA TCC TTG GAG GGT CCC AGG CCC TCA CCC ACA AGGTCC GTC 725 Val Ser Gly Ser Leu Glu Gly Pro Arg Pro Ser Pro Thr Arg SerVal 220 225 230 TCA ACA GCT GCA GGC CCT GAG GAC CAG CCC CTC ATG CCT ACAGGG TCA 773 Ser Thr Ala Ala Gly Pro Glu Asp Gln Pro Leu Met Pro Thr GlySer 235 240 245 GTC CCC CAC AGT GGT CTG AGA AGG CAC TGG GAG GTA CTG ATCGGG GTC 821 Val Pro His Ser Gly Leu Arg Arg His Trp Glu Val Leu Ile GlyVal 250 255 260 TTG GTG GTC TCC ATC CTG CTT CTC TCC CTC CTC CTC TTC CTCCTC CTC 869 Leu Val Val Ser Ile Leu Leu Leu Ser Leu Leu Leu Phe Leu LeuLeu 265 270 275 280 CAA CAC TGG CGT CAG GGA AAA CAC AGG ACA TTG GCC CAGAGA CAG GCT 917 Gln His Trp Arg Gln Gly Lys His Arg Thr Leu Ala Gln ArgGln Ala 285 290 295 GAT TTC CAA CGT CCT CCA GGG GCT GCC GAG CCA GAG CCCAAG GAC GGG 965 Asp Phe Gln Arg Pro Pro Gly Ala Ala Glu Pro Glu Pro LysAsp Gly 300 305 310 GGC CTA CAG AGG AGG TCC AGC CCA GCT GCT GAC GTC CAGGGA GAA AAC 1013 Gly Leu Gln Arg Arg Ser Ser Pro Ala Ala Asp Val Gln GlyGlu Asn 315 320 325 TTC TGT GCT GCC GTG AAG AAC ACA CAG CCT GAG GAC GGGGTG GAA ATG 1061 Phe Cys Ala Ala Val Lys Asn Thr Gln Pro Glu Asp Gly ValGlu Met 330 335 340 GAC ACT CGG CAG AGC CCA CAC GAT GAA GAC CCC CAG GCAGTG ACG TAT 1109 Asp Thr Arg Gln Ser Pro His Asp Glu Asp Pro Gln Ala ValThr Tyr 345 350 355 360 GCC AAG GTG AAA CAC TCC AGA CCT AGG AGA GAA ATGGCC TCT CCT CCC 1157 Ala Lys Val Lys His Ser Arg Pro Arg Arg Glu Met AlaSer Pro Pro 365 370 375 TCC CCA CTG TCT GGG GAA TTC CTG GAC ACA AAG GACAGA CAG GCA GAA 1205 Ser Pro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp ArgGln Ala Glu 380 385 390 GAG GAC AGA CAG ATG GAC ACT GAG GCT GCT GCA TCTGAA GCC CCC CAG 1253 Glu Asp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser GluAla Pro Gln 395 400 405 GAT GTG ACC TAC GCC CGG CTG CAC AGC TTT ACC CTCAGA CAG AAG GCA 1301 Asp Val Thr Tyr Ala Arg Leu His Ser Phe Thr Leu ArgGln Lys Ala 410 415 420 ACT GAG CCT CCT CCA TCC CAG GAA GGG GCC TCT CCAGCT GAG CCC AGT 1349 Thr Glu Pro Pro Pro Ser Gln Glu Gly Ala Ser Pro AlaGlu Pro Ser 425 430 435 440 GTC TAT GCC ACT CTG GCC ATC CAC TAATCCAGGGGGG ACCCAGACCC 1396 Val Tyr Ala Thr Leu Ala Ile His 445CACAAGCCAT GGAGACTCAG GACCCCAGAA GGCATGGAAG CTGCCTCCAG TAGACATCAC 1456TGAACCCCAG CCAGCCCAGA CCCCTGACAC AGACCACTAG AAGATTCCGG GAACGTTGGG 1516AGTCACCTGA TTCTGCAAAG ATAAATAATA TCCCTGCATT ATCAAAATAA AGTAGCAGAC 1576CTCTCAATTC ACAATGAGTT AACTGATAAA ACAAAACAGA AGTCAAAAA 1625 448 aminoacids amino acid linear protein 18 Met Ile Pro Thr Phe Thr Ala Leu LeuCys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr His Met Gln Ala GlyPro Leu Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro Gly Ser Val Ile SerTrp Gly Asn Ser Val Thr Ile Trp 35 40 45 Cys Gln Gly Thr Leu Glu Ala ArgGlu Tyr Arg Leu Asp Lys Glu Glu 50 55 60 Ser Pro Ala Pro Trp Asp Arg GlnAsn Pro Leu Glu Pro Lys Asn Lys 65 70 75 80 Ala Arg Phe Ser Ile Pro SerMet Thr Glu Asp Tyr Ala Gly Arg Tyr 85 90 95 Arg Cys Tyr Tyr Arg Ser ProVal Gly Trp Ser Gln Pro Ser Asp Pro 100 105 110 Leu Glu Leu Val Met ThrGly Ala Tyr Ser Lys Pro Thr Leu Ser Ala 115 120 125 Leu Pro Ser Pro LeuVal Thr Ser Gly Lys Ser Val Thr Leu Leu Cys 130 135 140 Gln Ser Arg SerPro Met Asp Thr Phe Leu Leu Ile Lys Glu Arg Ala 145 150 155 160 Ala HisPro Leu Leu His Leu Arg Ser Glu His Gly Ala Gln Gln His 165 170 175 GlnAla Glu Phe Pro Met Ser Pro Val Thr Ser Val His Gly Gly Thr 180 185 190Tyr Arg Cys Phe Ser Ser His Gly Phe Ser His Tyr Leu Leu Ser His 195 200205 Pro Ser Asp Pro Leu Glu Leu Ile Val Ser Gly Ser Leu Glu Gly Pro 210215 220 Arg Pro Ser Pro Thr Arg Ser Val Ser Thr Ala Ala Gly Pro Glu Asp225 230 235 240 Gln Pro Leu Met Pro Thr Gly Ser Val Pro His Ser Gly LeuArg Arg 245 250 255 His Trp Glu Val Leu Ile Gly Val Leu Val Val Ser IleLeu Leu Leu 260 265 270 Ser Leu Leu Leu Phe Leu Leu Leu Gln His Trp ArgGln Gly Lys His 275 280 285 Arg Thr Leu Ala Gln Arg Gln Ala Asp Phe GlnArg Pro Pro Gly Ala 290 295 300 Ala Glu Pro Glu Pro Lys Asp Gly Gly LeuGln Arg Arg Ser Ser Pro 305 310 315 320 Ala Ala Asp Val Gln Gly Glu AsnPhe Cys Ala Ala Val Lys Asn Thr 325 330 335 Gln Pro Glu Asp Gly Val GluMet Asp Thr Arg Gln Ser Pro His Asp 340 345 350 Glu Asp Pro Gln Ala ValThr Tyr Ala Lys Val Lys His Ser Arg Pro 355 360 365 Arg Arg Glu Met AlaSer Pro Pro Ser Pro Leu Ser Gly Glu Phe Leu 370 375 380 Asp Thr Lys AspArg Gln Ala Glu Glu Asp Arg Gln Met Asp Thr Glu 385 390 395 400 Ala AlaAla Ser Glu Ala Pro Gln Asp Val Thr Tyr Ala Arg Leu His 405 410 415 SerPhe Thr Leu Arg Gln Lys Ala Thr Glu Pro Pro Pro Ser Gln Glu 420 425 430Gly Ala Ser Pro Ala Glu Pro Ser Val Tyr Ala Thr Leu Ala Ile His 435 440445 2194 base pairs nucleic acid single linear cDNA pbm17 CDS 67..195919 TCTCTGTCCT GCCAGCACTG AGGGCTCATC CCTCTGCAGA GCGCGGGGTC ACCGGAAGGA 60GACGCC ATG ACG CCC GCC CTC ACA GCC CTG CTC TGC CTT GGG CTG AGT 108 MetThr Pro Ala Leu Thr Ala Leu Leu Cys Leu Gly Leu Ser 1 5 10 CTG GGC CCCAGG ACC CGC GTG CAG GCA GGG CCC TTC CCC AAA CCC ACC 156 Leu Gly Pro ArgThr Arg Val Gln Ala Gly Pro Phe Pro Lys Pro Thr 15 20 25 30 CTC TGG GCTGAG CCA GGC TCT GTG ATC AGC TGG GGG AGC CCC GTG ACC 204 Leu Trp Ala GluPro Gly Ser Val Ile Ser Trp Gly Ser Pro Val Thr 35 40 45 ATC TGG TGT CAGGGG AGC CTG GAG GCC CAG GAG TAC CAA CTG GAT AAA 252 Ile Trp Cys Gln GlySer Leu Glu Ala Gln Glu Tyr Gln Leu Asp Lys 50 55 60 GAG GGA AGC CCA GAGCCC TTG GAC AGA AAT AAC CCA CTG GAA CCC AAG 300 Glu Gly Ser Pro Glu ProLeu Asp Arg Asn Asn Pro Leu Glu Pro Lys 65 70 75 AAC AAG GCC AGA TTC TCCATC CCA TCC ATG ACA CAG CAC CAT GCA GGG 348 Asn Lys Ala Arg Phe Ser IlePro Ser Met Thr Gln His His Ala Gly 80 85 90 AGA TAC CGC TGC CAC TAT TACAGC TCT GCA GGC TGG TCA GAG CCC AGC 396 Arg Tyr Arg Cys His Tyr Tyr SerSer Ala Gly Trp Ser Glu Pro Ser 95 100 105 110 GAC CCC CTG GAG CTG GTGATG ACA GGA GCC TAT AGC AAA CCC ACC CTC 444 Asp Pro Leu Glu Leu Val MetThr Gly Ala Tyr Ser Lys Pro Thr Leu 115 120 125 TCA GCC CTG CCC AGC CCTGTG GTG GCC TCA GGG GGG AAT ATG ACC CTC 492 Ser Ala Leu Pro Ser Pro ValVal Ala Ser Gly Gly Asn Met Thr Leu 130 135 140 CGA TGT GGC TCA CAG AAGAGA TAT CAC CAT TTT GTT CTG ATG AAG GAA 540 Arg Cys Gly Ser Gln Lys ArgTyr His His Phe Val Leu Met Lys Glu 145 150 155 GGA GAA CAC CAG CTC CCCCGG ACC CTG GAC TCA CAG CAG CTC CAC AGT 588 Gly Glu His Gln Leu Pro ArgThr Leu Asp Ser Gln Gln Leu His Ser 160 165 170 GGG GGG TTC CAG GCC CTGTTC CCT GTG GGC CCC GTG AAC CCC AGC CAC 636 Gly Gly Phe Gln Ala Leu PhePro Val Gly Pro Val Asn Pro Ser His 175 180 185 190 AGG TGG AGG TTC ACATGC TAT TAC TAT TAT ATG AAC ACC CCC CGG GTG 684 Arg Trp Arg Phe Thr CysTyr Tyr Tyr Tyr Met Asn Thr Pro Arg Val 195 200 205 TGG TCC CAC CCC AGTGAC CCC CTG GAG ATT CTG CCC TCA GGC GTG TCT 732 Trp Ser His Pro Ser AspPro Leu Glu Ile Leu Pro Ser Gly Val Ser 210 215 220 AGG AAG CCC TCC CTCCTG ACC CTG CAG GGC CCT GTC CTG GCC CCT GGG 780 Arg Lys Pro Ser Leu LeuThr Leu Gln Gly Pro Val Leu Ala Pro Gly 225 230 235 CAG AGT CTG ACC CTCCAG TGT GGC TCT GAT GTC GGC TAC GAC AGA TTT 828 Gln Ser Leu Thr Leu GlnCys Gly Ser Asp Val Gly Tyr Asp Arg Phe 240 245 250 GTT CTG TAT AAG GAGGGG GAA CGT GAC TTC CTC CAG CGC CCT GGC CAG 876 Val Leu Tyr Lys Glu GlyGlu Arg Asp Phe Leu Gln Arg Pro Gly Gln 255 260 265 270 CAG CCC CAG GCTGGG CTC TCC CAG GCC AAC TTC ACC CTG GGC CCT GTG 924 Gln Pro Gln Ala GlyLeu Ser Gln Ala Asn Phe Thr Leu Gly Pro Val 275 280 285 AGC CCC TCC AATGGG GGC CAG TAC AGG TGC TAC GGT GCA CAC AAC CTC 972 Ser Pro Ser Asn GlyGly Gln Tyr Arg Cys Tyr Gly Ala His Asn Leu 290 295 300 TCC TCC GAG TGGTCG GCC CCC AGC GAC CCC CTG AAC ATC CTG ATG GCA 1020 Ser Ser Glu Trp SerAla Pro Ser Asp Pro Leu Asn Ile Leu Met Ala 305 310 315 GGA CAG ATC TATGAC ACC GTC TCC CTG TCA GCA CAG CCG GGC CCC ACA 1068 Gly Gln Ile Tyr AspThr Val Ser Leu Ser Ala Gln Pro Gly Pro Thr 320 325 330 GTG GCC TCA GGAGAG AAC GTG ACC CTG CTG TGT CAG TCA TGG TGG CAG 1116 Val Ala Ser Gly GluAsn Val Thr Leu Leu Cys Gln Ser Trp Trp Gln 335 340 345 350 TTT GAC ACTTTC CTT CTG ACC AAA GAA GGG GCA GCC CAT CCC CCA CTG 1164 Phe Asp Thr PheLeu Leu Thr Lys Glu Gly Ala Ala His Pro Pro Leu 355 360 365 CGT CTG AGATCA ATG TAC GGA GCT CAT AAG TAC CAG GCT GAA TTC CCC 1212 Arg Leu Arg SerMet Tyr Gly Ala His Lys Tyr Gln Ala Glu Phe Pro 370 375 380 ATG AGT CCTGTG ACC TCA GCC CAC GCG GGG ACC TAC AGG TGC TAC GGC 1260 Met Ser Pro ValThr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly 385 390 395 TCA CGC AGCTCC AAC CCC TAC CTG CTG TCT CAC CCC AGT GAG CCC CTG 1308 Ser Arg Ser SerAsn Pro Tyr Leu Leu Ser His Pro Ser Glu Pro Leu 400 405 410 GAG CTC GTGGTC TCA GGA CAC TCT GGA GGC TCC AGC CTC CCA CCC ACA 1356 Glu Leu Val ValSer Gly His Ser Gly Gly Ser Ser Leu Pro Pro Thr 415 420 425 430 GGG CCGCCC TCC ACA CCT GGT CTG GGA AGA TAC CTG GAG GTT TTG ATT 1404 Gly Pro ProSer Thr Pro Gly Leu Gly Arg Tyr Leu Glu Val Leu Ile 435 440 445 GGG GTCTCG GTG GCC TTC GTC CTG CTG CTC TTC CTC CTC CTC TTC CTC 1452 Gly Val SerVal Ala Phe Val Leu Leu Leu Phe Leu Leu Leu Phe Leu 450 455 460 CTC CTCCGA CGT CAG CGT CAC AGC AAA CAC AGG ACA TCT GAC CAG AGA 1500 Leu Leu ArgArg Gln Arg His Ser Lys His Arg Thr Ser Asp Gln Arg 465 470 475 AAG ACTGAT TTC CAG CGT CCT GCA GGG GCT GCG GAG ACA GAG CCC AAG 1548 Lys Thr AspPhe Gln Arg Pro Ala Gly Ala Ala Glu Thr Glu Pro Lys 480 485 490 GAC AGGGGC CTG CTG AGG AGG TCC AGC CCA GCT GCT GAC GTC CAG GAA 1596 Asp Arg GlyLeu Leu Arg Arg Ser Ser Pro Ala Ala Asp Val Gln Glu 495 500 505 510 GAAAAC CTC TAT GCT GCC GTG AAG GAC ACA CAG TCT GAG GAC GGG GTG 1644 Glu AsnLeu Tyr Ala Ala Val Lys Asp Thr Gln Ser Glu Asp Gly Val 515 520 525 GAGCTG GAC AGT CAG AGC CCA CAC GAT GAA GAC CCC CAC GCA GTG ACG 1692 Glu LeuAsp Ser Gln Ser Pro His Asp Glu Asp Pro His Ala Val Thr 530 535 540 TATGCC CCG GTG AAA CAC TCC AGT CCT AGG AGA GAA ATG GCC TCT CCT 1740 Tyr AlaPro Val Lys His Ser Ser Pro Arg Arg Glu Met Ala Ser Pro 545 550 555 CCTTCC CCA CTG TCT GGG GAA TTC CTG GAC ACA AAG GAC AGA CAG GCA 1788 Pro SerPro Leu Ser Gly Glu Phe Leu Asp Thr Lys Asp Arg Gln Ala 560 565 570 GAAGAG GAC AGA CAG ATG GAC ACT GAG GCT GCT GCA TCT GAA GCC TCC 1836 Glu GluAsp Arg Gln Met Asp Thr Glu Ala Ala Ala Ser Glu Ala Ser 575 580 585 590CAG GAT GTG ACC TAC GCC CAG CTG CAC AGC TTG ACC CTT AGA CGG AAG 1884 GlnAsp Val Thr Tyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Lys 595 600 605GCA ACT GAG CCT CCT CCA TCC CAG GAA GGG GAA CCT CCA GCT GAG CCC 1932 AlaThr Glu Pro Pro Pro Ser Gln Glu Gly Glu Pro Pro Ala Glu Pro 610 615 620AGC ATC TAC GCC ACT CTG GCC ATC CAC TAG CCCGGGGGGT ACGCAGACCC 1982 SerIle Tyr Ala Thr Leu Ala Ile His 625 630 CACACTCAGC AGAAGGAGAC TCAGGACTGCTGAAGGACGG GAGCTGCCCC CAGTGGACAC 2042 CAGTGAACCC CAGTCAGCCT GGACCCCTAACACAGACCAT GAGGAGACGC TGGGAACTTG 2102 TGGGACTCAC CTGACTCAAA GATGACTAATATCGTCCCAT TTTGGAAATA AAGCAACAGA 2162 CTTCTCAAGC AGGTCGTCTC GTTCCAAGATCT 2194 631 amino acids amino acid linear protein 20 Met Thr Pro Ala LeuThr Ala Leu Leu Cys Leu Gly Leu Ser Leu Gly 1 5 10 15 Pro Arg Thr ArgVal Gln Ala Gly Pro Phe Pro Lys Pro Thr Leu Trp 20 25 30 Ala Glu Pro GlySer Val Ile Ser Trp Gly Ser Pro Val Thr Ile Trp 35 40 45 Cys Gln Gly SerLeu Glu Ala Gln Glu Tyr Gln Leu Asp Lys Glu Gly 50 55 60 Ser Pro Glu ProLeu Asp Arg Asn Asn Pro Leu Glu Pro Lys Asn Lys 65 70 75 80 Ala Arg PheSer Ile Pro Ser Met Thr Gln His His Ala Gly Arg Tyr 85 90 95 Arg Cys HisTyr Tyr Ser Ser Ala Gly Trp Ser Glu Pro Ser Asp Pro 100 105 110 Leu GluLeu Val Met Thr Gly Ala Tyr Ser Lys Pro Thr Leu Ser Ala 115 120 125 LeuPro Ser Pro Val Val Ala Ser Gly Gly Asn Met Thr Leu Arg Cys 130 135 140Gly Ser Gln Lys Arg Tyr His His Phe Val Leu Met Lys Glu Gly Glu 145 150155 160 His Gln Leu Pro Arg Thr Leu Asp Ser Gln Gln Leu His Ser Gly Gly165 170 175 Phe Gln Ala Leu Phe Pro Val Gly Pro Val Asn Pro Ser His ArgTrp 180 185 190 Arg Phe Thr Cys Tyr Tyr Tyr Tyr Met Asn Thr Pro Arg ValTrp Ser 195 200 205 His Pro Ser Asp Pro Leu Glu Ile Leu Pro Ser Gly ValSer Arg Lys 210 215 220 Pro Ser Leu Leu Thr Leu Gln Gly Pro Val Leu AlaPro Gly Gln Ser 225 230 235 240 Leu Thr Leu Gln Cys Gly Ser Asp Val GlyTyr Asp Arg Phe Val Leu 245 250 255 Tyr Lys Glu Gly Glu Arg Asp Phe LeuGln Arg Pro Gly Gln Gln Pro 260 265 270 Gln Ala Gly Leu Ser Gln Ala AsnPhe Thr Leu Gly Pro Val Ser Pro 275 280 285 Ser Asn Gly Gly Gln Tyr ArgCys Tyr Gly Ala His Asn Leu Ser Ser 290 295 300 Glu Trp Ser Ala Pro SerAsp Pro Leu Asn Ile Leu Met Ala Gly Gln 305 310 315 320 Ile Tyr Asp ThrVal Ser Leu Ser Ala Gln Pro Gly Pro Thr Val Ala 325 330 335 Ser Gly GluAsn Val Thr Leu Leu Cys Gln Ser Trp Trp Gln Phe Asp 340 345 350 Thr PheLeu Leu Thr Lys Glu Gly Ala Ala His Pro Pro Leu Arg Leu 355 360 365 ArgSer Met Tyr Gly Ala His Lys Tyr Gln Ala Glu Phe Pro Met Ser 370 375 380Pro Val Thr Ser Ala His Ala Gly Thr Tyr Arg Cys Tyr Gly Ser Arg 385 390395 400 Ser Ser Asn Pro Tyr Leu Leu Ser His Pro Ser Glu Pro Leu Glu Leu405 410 415 Val Val Ser Gly His Ser Gly Gly Ser Ser Leu Pro Pro Thr GlyPro 420 425 430 Pro Ser Thr Pro Gly Leu Gly Arg Tyr Leu Glu Val Leu IleGly Val 435 440 445 Ser Val Ala Phe Val Leu Leu Leu Phe Leu Leu Leu PheLeu Leu Leu 450 455 460 Arg Arg Gln Arg His Ser Lys His Arg Thr Ser AspGln Arg Lys Thr 465 470 475 480 Asp Phe Gln Arg Pro Ala Gly Ala Ala GluThr Glu Pro Lys Asp Arg 485 490 495 Gly Leu Leu Arg Arg Ser Ser Pro AlaAla Asp Val Gln Glu Glu Asn 500 505 510 Leu Tyr Ala Ala Val Lys Asp ThrGln Ser Glu Asp Gly Val Glu Leu 515 520 525 Asp Ser Gln Ser Pro His AspGlu Asp Pro His Ala Val Thr Tyr Ala 530 535 540 Pro Val Lys His Ser SerPro Arg Arg Glu Met Ala Ser Pro Pro Ser 545 550 555 560 Pro Leu Ser GlyGlu Phe Leu Asp Thr Lys Asp Arg Gln Ala Glu Glu 565 570 575 Asp Arg GlnMet Asp Thr Glu Ala Ala Ala Ser Glu Ala Ser Gln Asp 580 585 590 Val ThrTyr Ala Gln Leu His Ser Leu Thr Leu Arg Arg Lys Ala Thr 595 600 605 GluPro Pro Pro Ser Gln Glu Gly Glu Pro Pro Ala Glu Pro Ser Ile 610 615 620Tyr Ala Thr Leu Ala Ile His 625 630 2061 base pairs nucleic acid singlelinear cDNA pbmnew CDS 67..1833 21 TTTGTGTCCT GCCAGGCACC GTGGTCTCATCCGCCTGCAC AGCTGAGTCC AGTGGGAGCT 60 GACGCC ATG ACC CTC ACC CTC TCA GTCCTG ATT TGC CTC GGG CTG AGT 108 Met Thr Leu Thr Leu Ser Val Leu Ile CysLeu Gly Leu Ser 1 5 10 GTG GGC CCC AGG ACC TGC GTG CAG GCA GGC ACC CTCCCC AAA CCC ACC 156 Val Gly Pro Arg Thr Cys Val Gln Ala Gly Thr Leu ProLys Pro Thr 15 20 25 30 CTC TGG GCT GAG CCA GCC TCT GTG ATA GCT CGG GGGAAG CCC GTG ACC 204 Leu Trp Ala Glu Pro Ala Ser Val Ile Ala Arg Gly LysPro Val Thr 35 40 45 CTC TGG TGT CAG GGG CCC CTG GAG ACT GAG GAG TAC CGTCTG GAT AAG 252 Leu Trp Cys Gln Gly Pro Leu Glu Thr Glu Glu Tyr Arg LeuAsp Lys 50 55 60 GAG GGA CTC CCA TGG GCC CGG AAG AGA CAG AAC CCA CTG GAGCCT GGA 300 Glu Gly Leu Pro Trp Ala Arg Lys Arg Gln Asn Pro Leu Glu ProGly 65 70 75 GCC AAG GCC AAG TTC CAC ATT CCA TCC ACG GTG TAT GAC AGT GCAGGG 348 Ala Lys Ala Lys Phe His Ile Pro Ser Thr Val Tyr Asp Ser Ala Gly80 85 90 CGA TAC CGC TGC TAC TAT GAG ACC CCT GCA GGC TGG TCA GAG CCC AGT396 Arg Tyr Arg Cys Tyr Tyr Glu Thr Pro Ala Gly Trp Ser Glu Pro Ser 95100 105 110 GAC CCC CTG GAG CTG GTG GCG ACA GGA TTC TAT GCA GAA CCC ACTCTT 444 Asp Pro Leu Glu Leu Val Ala Thr Gly Phe Tyr Ala Glu Pro Thr Leu115 120 125 TTA GCC CTG CCG AGT CCT GTG GTG GCC TCA GGA GGA AAT GTG ACCCTC 492 Leu Ala Leu Pro Ser Pro Val Val Ala Ser Gly Gly Asn Val Thr Leu130 135 140 CAG TGT GAT ACA CTG GAC GGA CTT CTC ACG TTT GTT CTT GTT GAGGAA 540 Gln Cys Asp Thr Leu Asp Gly Leu Leu Thr Phe Val Leu Val Glu Glu145 150 155 GAA CAG AAG CTC CCC AGG ACC CTG TAC TCA CAG AAG CTC CCC AAAGGG 588 Glu Gln Lys Leu Pro Arg Thr Leu Tyr Ser Gln Lys Leu Pro Lys Gly160 165 170 CCA TCC CAG GCC CTG TTC CCT GTG GGT CCC GTG ACC CCC AGC TGCAGG 636 Pro Ser Gln Ala Leu Phe Pro Val Gly Pro Val Thr Pro Ser Cys Arg175 180 185 190 TGG AGG TTC AGA TGC TAT TAC TAT TAC AGG AAA AAC CCT CAGGTG TGG 684 Trp Arg Phe Arg Cys Tyr Tyr Tyr Tyr Arg Lys Asn Pro Gln ValTrp 195 200 205 TCG AAC CCC AGT GAC CTC CTG GAG ATT CTG GTC CCA GGC GTGTCT AGG 732 Ser Asn Pro Ser Asp Leu Leu Glu Ile Leu Val Pro Gly Val SerArg 210 215 220 AAG CCC TCC CTC CTG ATC CCG CAG GGC TCT GTC GTG GCC CGCGGA GGC 780 Lys Pro Ser Leu Leu Ile Pro Gln Gly Ser Val Val Ala Arg GlyGly 225 230 235 AGC CTG ACC CTG CAG TGT CGC TCT GAT GTC GGC TAT GAC ATATTC GTT 828 Ser Leu Thr Leu Gln Cys Arg Ser Asp Val Gly Tyr Asp Ile PheVal 240 245 250 CTG TAC AAG GAG GGG GAA CAT GAC CTC GTC CAG GGC TCT GGCCAG CAG 876 Leu Tyr Lys Glu Gly Glu His Asp Leu Val Gln Gly Ser Gly GlnGln 255 260 265 270 CCC CAG GCT GGG CTC TCC CAG GCC AAC TTC ACC CTG GGCCCT GTG AGC 924 Pro Gln Ala Gly Leu Ser Gln Ala Asn Phe Thr Leu Gly ProVal Ser 275 280 285 CGC TCC CAC GGG GGC CAG TAC AGA TGC TAC GGT GCA CACAAC CTC TCC 972 Arg Ser His Gly Gly Gln Tyr Arg Cys Tyr Gly Ala His AsnLeu Ser 290 295 300 CCT AGG TGG TCG GCC CCC AGC GAC CCC CTG GAC ATC CTGATC GCA GGA 1020 Pro Arg Trp Ser Ala Pro Ser Asp Pro Leu Asp Ile Leu IleAla Gly 305 310 315 CTG ATC CCT GAC ATA CCC GCC CTC TCG GTG CAG CCG GGCCCC AAG GTG 1068 Leu Ile Pro Asp Ile Pro Ala Leu Ser Val Gln Pro Gly ProLys Val 320 325 330 GCC TCA GGA GAG AAC GTG ACC CTG CTG TGT CAG TCA TGGCAT CAG ATA 1116 Ala Ser Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp HisGln Ile 335 340 345 350 GAC ACT TTC TTT TTG ACC AAG GAG GGG GCA GCC CATCCC CCG CTG TGT 1164 Asp Thr Phe Phe Leu Thr Lys Glu Gly Ala Ala His ProPro Leu Cys 355 360 365 CTA AAG TCA AAG TAC CAG TCT TAT AGA CAC CAG GCTGAA TTC TCC ATG 1212 Leu Lys Ser Lys Tyr Gln Ser Tyr Arg His Gln Ala GluPhe Ser Met 370 375 380 AGT CCT GTG ACC TCA GCC CAG GGT GGA ACC TAC CGATGC TAC AGC GCA 1260 Ser Pro Val Thr Ser Ala Gln Gly Gly Thr Tyr Arg CysTyr Ser Ala 385 390 395 ATC AGG TCC TAC CCC TAC CTG CTG TCC AGC CCT AGTTAC CCC CAG GAG 1308 Ile Arg Ser Tyr Pro Tyr Leu Leu Ser Ser Pro Ser TyrPro Gln Glu 400 405 410 CTC GTG GTC TCA GGA CCC TCT GGG GAT CCC AGC CTCTCA CCT ACA GGC 1356 Leu Val Val Ser Gly Pro Ser Gly Asp Pro Ser Leu SerPro Thr Gly 415 420 425 430 TCC ACC CCC ACA CCT GGC CCT GAG GAC CAG CCCCTC ACC CCC ACG GGG 1404 Ser Thr Pro Thr Pro Gly Pro Glu Asp Gln Pro LeuThr Pro Thr Gly 435 440 445 TTG GAT CCC CAG AGT GGT CTG GGA AGG CAC CTGGGG GTT GTG ACT GGG 1452 Leu Asp Pro Gln Ser Gly Leu Gly Arg His Leu GlyVal Val Thr Gly 450 455 460 GTC TCA GTG GCC TTC GTC CTG CTG CTG TTC CTCCTC CTC TTC CTC CTC 1500 Val Ser Val Ala Phe Val Leu Leu Leu Phe Leu LeuLeu Phe Leu Leu 465 470 475 CTC CGA CAT CGG CAT CAG AGC AAA CAC AGG ACATCG GCC CAT TTC TAC 1548 Leu Arg His Arg His Gln Ser Lys His Arg Thr SerAla His Phe Tyr 480 485 490 CGT CCT GCA GGG GCT GCG GGG CCA GAG CCC AAGGAC CAG GGC CTG CAG 1596 Arg Pro Ala Gly Ala Ala Gly Pro Glu Pro Lys AspGln Gly Leu Gln 495 500 505 510 AAG AGG GCC AGC CCA GTT GCT GAC ATC CAGGAG GAA ATT CTC AAT GCT 1644 Lys Arg Ala Ser Pro Val Ala Asp Ile Gln GluGlu Ile Leu Asn Ala 515 520 525 GCC GTG AAG GAC ACA CAG CCC AAG GAC GGGGTG GAG ATG GAT GCT CGG 1692 Ala Val Lys Asp Thr Gln Pro Lys Asp Gly ValGlu Met Asp Ala Arg 530 535 540 GCT GCT GCA TCT GAA GCC CCC CAG GAT GTGACC TAC GCC CAG CTG CAC 1740 Ala Ala Ala Ser Glu Ala Pro Gln Asp Val ThrTyr Ala Gln Leu His 545 550 555 AGC TTG ACC CTC AGA CGG GAG GCA ACT GAGCCT CCT CCA TCC CAG GAA 1788 Ser Leu Thr Leu Arg Arg Glu Ala Thr Glu ProPro Pro Ser Gln Glu 560 565 570 AGG GAA CCT CCA GCT GAA CCC AGC ATC TACGCC CCC CTG GCC ATC CAC 1836 Arg Glu Pro Pro Ala Glu Pro Ser Ile Tyr AlaPro Leu Ala Ile His 575 580 585 590 TAG CCCACGGGGG ACCCAGATCT CATACTCAACAGAAGGAGAC TCAGAGACTC 1889 CAGAAGGCAC AGGAGCTGCC CCCAGTGGAC ACCAATGAACCCCAGCCAGC CTGGACCCCT 1949 AACAAAGACC ACCAGGACAT CCTGGGAACT CTGGGACTCACTAGATTCTG CAGTCAAAGA 2009 TGACTAATAT CCTTGCATTT TTGAAATGAA GCCACAGACTTCTCAATAAA TC 2061 590 amino acids amino acid linear protein 22 Met ThrLeu Thr Leu Ser Val Leu Ile Cys Leu Gly Leu Ser Val Gly 1 5 10 15 ProArg Thr Cys Val Gln Ala Gly Thr Leu Pro Lys Pro Thr Leu Trp 20 25 30 AlaGlu Pro Ala Ser Val Ile Ala Arg Gly Lys Pro Val Thr Leu Trp 35 40 45 CysGln Gly Pro Leu Glu Thr Glu Glu Tyr Arg Leu Asp Lys Glu Gly 50 55 60 LeuPro Trp Ala Arg Lys Arg Gln Asn Pro Leu Glu Pro Gly Ala Lys 65 70 75 80Ala Lys Phe His Ile Pro Ser Thr Val Tyr Asp Ser Ala Gly Arg Tyr 85 90 95Arg Cys Tyr Tyr Glu Thr Pro Ala Gly Trp Ser Glu Pro Ser Asp Pro 100 105110 Leu Glu Leu Val Ala Thr Gly Phe Tyr Ala Glu Pro Thr Leu Leu Ala 115120 125 Leu Pro Ser Pro Val Val Ala Ser Gly Gly Asn Val Thr Leu Gln Cys130 135 140 Asp Thr Leu Asp Gly Leu Leu Thr Phe Val Leu Val Glu Glu GluGln 145 150 155 160 Lys Leu Pro Arg Thr Leu Tyr Ser Gln Lys Leu Pro LysGly Pro Ser 165 170 175 Gln Ala Leu Phe Pro Val Gly Pro Val Thr Pro SerCys Arg Trp Arg 180 185 190 Phe Arg Cys Tyr Tyr Tyr Tyr Arg Lys Asn ProGln Val Trp Ser Asn 195 200 205 Pro Ser Asp Leu Leu Glu Ile Leu Val ProGly Val Ser Arg Lys Pro 210 215 220 Ser Leu Leu Ile Pro Gln Gly Ser ValVal Ala Arg Gly Gly Ser Leu 225 230 235 240 Thr Leu Gln Cys Arg Ser AspVal Gly Tyr Asp Ile Phe Val Leu Tyr 245 250 255 Lys Glu Gly Glu His AspLeu Val Gln Gly Ser Gly Gln Gln Pro Gln 260 265 270 Ala Gly Leu Ser GlnAla Asn Phe Thr Leu Gly Pro Val Ser Arg Ser 275 280 285 His Gly Gly GlnTyr Arg Cys Tyr Gly Ala His Asn Leu Ser Pro Arg 290 295 300 Trp Ser AlaPro Ser Asp Pro Leu Asp Ile Leu Ile Ala Gly Leu Ile 305 310 315 320 ProAsp Ile Pro Ala Leu Ser Val Gln Pro Gly Pro Lys Val Ala Ser 325 330 335Gly Glu Asn Val Thr Leu Leu Cys Gln Ser Trp His Gln Ile Asp Thr 340 345350 Phe Phe Leu Thr Lys Glu Gly Ala Ala His Pro Pro Leu Cys Leu Lys 355360 365 Ser Lys Tyr Gln Ser Tyr Arg His Gln Ala Glu Phe Ser Met Ser Pro370 375 380 Val Thr Ser Ala Gln Gly Gly Thr Tyr Arg Cys Tyr Ser Ala IleArg 385 390 395 400 Ser Tyr Pro Tyr Leu Leu Ser Ser Pro Ser Tyr Pro GlnGlu Leu Val 405 410 415 Val Ser Gly Pro Ser Gly Asp Pro Ser Leu Ser ProThr Gly Ser Thr 420 425 430 Pro Thr Pro Gly Pro Glu Asp Gln Pro Leu ThrPro Thr Gly Leu Asp 435 440 445 Pro Gln Ser Gly Leu Gly Arg His Leu GlyVal Val Thr Gly Val Ser 450 455 460 Val Ala Phe Val Leu Leu Leu Phe LeuLeu Leu Phe Leu Leu Leu Arg 465 470 475 480 His Arg His Gln Ser Lys HisArg Thr Ser Ala His Phe Tyr Arg Pro 485 490 495 Ala Gly Ala Ala Gly ProGlu Pro Lys Asp Gln Gly Leu Gln Lys Arg 500 505 510 Ala Ser Pro Val AlaAsp Ile Gln Glu Glu Ile Leu Asn Ala Ala Val 515 520 525 Lys Asp Thr GlnPro Lys Asp Gly Val Glu Met Asp Ala Arg Ala Ala 530 535 540 Ala Ser GluAla Pro Gln Asp Val Thr Tyr Ala Gln Leu His Ser Leu 545 550 555 560 ThrLeu Arg Arg Glu Ala Thr Glu Pro Pro Pro Ser Gln Glu Arg Glu 565 570 575Pro Pro Ala Glu Pro Ser Ile Tyr Ala Pro Leu Ala Ile His 580 585 590 28base pairs nucleic acid single linear Probe 23 TATGCGGCCG CCATGATGACAATGTGGT 28 25 base pairs nucleic acid single linear Probe 24 TATGCGGCCGCCCCTTGCGA TAGCG 25 31 base pairs nucleic acid single linearOligonucleotide 25 ATAGTCGACA ACGCCATCAT GAGATGTGGT G 31 29 Nucleotidesnucleic acid single linear Oligonucleotide 26 TAAAGATCTG GGCTCGTTAGCTGTCGGGT 29 33 nucleotides nucleic acid single linear Oligonucleotide27 TATAGATCTA CCCCCAGGTG CCTTCCCAGA CCA 33 42 amino acids amino acidlinear peptide 28 Leu Xaa Leu Ser Xaa Xaa Pro Arg Thr Xaa Xaa Gln XaaGly Xaa Xaa Pro 5 10 15 Xaa Pro Thr Leu Trp Ala Glu Pro Xaa Ser Phe IleXaa Xaa Ser Asp Pro 20 25 30 Lys Leu Xaa Leu Val Xaa Thr Gly 35 40 8amino acids amino acid linear peptide 29 Asp Tyr Lys Asp Asp Asp Asp Lys5

What is claimed is:
 1. An isolated DNA encoding an LIR polypeptide,wherein said LIR polypeptide comprises amino acid sequences selectedfrom the group consisting of: a) amino acids 5 to 50 of SEQ ID NO:2; andb) an amino acid sequence that is at least 77% identical to the sequenceof a).
 2. DNA encoding an LIR polypeptide, wherein said LIR polypeptidecomprises the amino acid sequence: Leu Xaa_(a) Leu Ser Xaa_(b) Xaa_(c)Pro Arg Thr Xaa_(d) Xaa_(e) Gln Xaa_(f) Gly Xaa_(g) Xaa_(h) Pro Xaa_(i)Pro Thr Leu Trp Ala Glu Pro Xaa_(j) Ser Phe Ile Xaa_(j) Xaa₇₀ Ser AspPro Lys Leu Xaa_(k) Leu Val Xaa_(m) Thr Gly where Xaa_(a) is Gly or Arg;Xaa_(b) is Leu or Val; Xaa_(c) is Gly or Asp; Xaa_(d) is His Arg or Cys;Xaa_(e) is Val or Met; Xaa_(f) is Ala or Thr; Xaa_(g) is His Pro or Thr,Xaa_(h) Leu Ile or Phe; Xaa_(i) is Gly Asp or Ala; Xaa_(j) is Thr IleSer or Ala; Xaa_(k) is Gly or Val; Xaa_(m) is Met or Ala; and Xaa₇₀ is asequence of 70 amino acids.
 3. DNA encoding an LIR polypeptide, whereinsaid LIR polypeptide is encoded by DNA selected from the groupconsisting of: a) DNA capable of hybridizing under highly stringentconditions to a probe consisting essentially of nucleotides 310 to 1684of SEQ ID NO:1; and b) DNA capable of hybridizing under highly stringentconditions to DNA complementary to the probe of a), wherein the highlystringent conditions comprise a hybridizing temperature of at least 63°C.
 4. An isolated DNA encoding an LIR polypeptide, wherein said LIRpolypeptide comprises an amino acid sequence that is at least 90%identical to an amino acid sequence selected from the group consistingof: a) SEQ ID NO:2; b) SEQ ID NO:4; c) SEQ ID NO:8; d) SEQ ID NO:10; e)SEQ ID NO:12; f) SEQ ID NO:14; g) SEQ ED NO:16; h) SEQ ID NO:18; i) SEQD NO:20; and j) SEQ ID NO:22.
 5. An isolated DNA of claim 4, whereinsaid LIR polypeptide comprises an amino acid sequence selected from thegroup consisting of: a) SEQ ID NO:2; b) SEQ ID NO:4; c) SEQ ID NO:8; d)SEQ ID NO:10; e) SEQ ID NO:12; f) SEQ ID NO:14; g) SEQ ID NO:16; h) SEQID NO:18; i) SEQ ID NO:20; and j) SEQ ID NO:22.
 6. An isolated DNAencoding a soluble LIR polypeptide, wherein said soluble LIR polypeptidecomprises an amino acid sequence that is at least 90% identical to asequence selected from the group consisting of: a) the extracellulardomain of LIR family members comprising sequences selected from thegroup consisting of: amino acids x₁ to 458 of SEQ ID NO:2, wherein x₁ isamino acid 1 or 17; amino acids x₂ to 459 of SEQ ID NO:4, wherein x₂ isamino acid 1 or 17; amino acids x₃ to 439 of SEQ ID NO:8, wherein x₃ isamino acids 1 or 17; amino acids x₄ to 458 of SEQ ID NO:10, wherein x₄is amino acid 1 or 17; amino acids x₅ to 241 of SEQ ID NO:12, wherein x₅is amino acid 1 or 17; amino acids x₆ to 461 of SEQ ID NO:14, wherein x₆is amino acid 1 or 17; amino acids x₇ to 449 of SEQ ED NO:16, wherein x₇is amino acid 1 or 17; amino acids x₈ to 259 of SEQ ID NO:18, wherein x₈is amino acid 1 or 17; amino acids x₉ to 443 of SEQ ID NO:20, wherein x₉is amino acid 1 or 17; and, amino acids x₁₀ to 456 of SEQ ID NO:22,wherein x₁₀ is amino acid 1 or 17; b) a fragment of any of the LIRextracellular domains of a), wherein said soluble LIR polypeptide bindsan MHC molecule.
 7. A DNA of claim 6, wherein said soluble LIRpolypeptide comprises an amino acid sequence selected from the groupconsisting of: a) the extracellular domain of LIR family memberscomprising sequences selected from the group consisting of: amino acidsx₁ to 458 of SEQ ID NO:2, wherein x₁ is amino acid 1 or 17; amino acidsx₂ to 459 of SEQ ID NO:4, wherein x₂ is amino acid 1 or 17; amino acidsx₃ to 439 of SEQ ID NO:8, wherein x₃ is amino acids 1 or 17; amino acidsx₄ to 458 of SEQ ID NO:10, wherein x₄ is amino acid 1 or 17; amino acidsx₅ to 241 of SEQ ID NO:12, wherein x₅ is amino acid 1 or 17; amino acidsx₆ to 461 of SEQ ID NO:14, wherein x₆ is amino acid 1 or 17; amino acidsx₇ to 449 of SEQ ID NO:16, wherein x₇ is amino acid 1 or 17;. aminoacids x₈ to 259 of SEQ ID NO:18, wherein x₈ is amino acid 1 or 17; aminoacids x₉ to 443 of SEQ ID NO:20, wherein x₉ is amino acid 1 or 17; and,amino acids x₁₀ to 456 of SEQ ID NO:22, wherein x₁₀ is amino acid 1 or17; b) a fragment of any of the LIR extracellular domains of a).
 8. AnLIR polypeptide comprising an amino acid sequence selected from thegroup consisting of: a) amino acids 5 to 50 of SEQ ID NO:2; and b) anamino acid sequence that is at least 77% identical to the sequence ofa).
 9. An LIR polypeptide comprising the amino acid sequence: LeuXaa_(a) Leu Ser Xaa_(b) Xaa_(c) Pro Arg Thr Xaa_(d) Xaa_(e) Gln Xaa_(f)Gly Xaa_(g) Xaa_(h) Pro Xaa_(i) Pro Thr Leu Trp Ala Glu Pro Xaa_(j) SerPhe Ile Xaa_(j) Xaa₇₀ Ser Asp Pro Lys Leu Xaa_(k) Leu Val Xaa_(m) ThrGly where Xaa_(a) is Gly or Arg; Xaa_(b) is Leu or Val; Xaa_(c) is Glyor Asp; Xaa_(d) is His Arg or Cys; Xaa_(e) is Val or Met; Xaa_(f) is Alaor Thr, Xaa_(g) is His Pro or Thr, Xaa_(h) Leu Ile or Phe; Xaa_(i) isGly Asp or Ala; Xaa_(j) is Thr Ile Ser or Ala; Xaa_(k) is Gly or Val;Xaa_(m) is Met or Ala; and Xaa₇₀ is a sequence of 70 amino acids.
 10. Anisolated polypeptide encoded by DNA selected from the group consistingof: a) DNA capable of hybridizing under highly stringent conditions to aprobe consisting essentially of nucleotides 310 to 1684 of SEQ ID NO:1;and b) DNA capable of hybridizing under highly stringent conditions toDNA complementary to the probe of a), wherein the highly stringentconditions comprise a hybridizing temperature of at least 63° C.
 11. Asoluble LIR polypeptide comprising an amino acid sequence that is atleast 90% identical to a sequence selected from the group consisting of:a) the extracellular domain of LIR family members, the extracellulardomains selected from the group consisting of amino acids x₁ to 458 ofSEQ ID NO:2, wherein x₁ is amino acid 1 or 17; amino acids x₂ to 458 ofSEQ ID NO:4, wherein x₂ is amino acid 1 or 17; amino acids x₃ to 439 ofSEQ ID NO:8, wherein x₃ is amino acid 1 or 17; amino acids x₄ to 458 ofSEQ ID NO:10, wherein x₄ is amino acid 1 or 17, amino acids x₅ to 242 ofSEQ ID NO:12, wherein x₅ is amino acid 1 or 17; amino acids x₆ to 461 ofSEQ ID NO:14, wherein x₆ is amino acid 1 or 17; amino acids x₇ to 449 ofSEQ ID NO:16, wherein x₇ is amino acid 1 or 17; amino acids x₈ to 259 ofSEQ ID NO:18, wherein x₈ is amino acid 1 or 17; amino acids x₉ to 443 ofSEQ ID NO:20, wherein x₉ is amino acid 1 or 17; and, amino acids x₁₀ to456 of SEQ ID NO:22, wherein x₁₀ is amino acid 1 or 17; b) a fragment ofany of the LIR extracellular domains of a), wherein the fragment iscapable of binding a ligand.
 12. A soluble polypeptide of claim 11comprising an amino acid sequence selected from the group consisting of:a) the extracellular domain of LIR family members, the extracellulardomains selected from the group consisting of amino acids x₁ to 458 ofSEQ ID NO:2, wherein x₁ is amino acid 1 or 17; amino acids x₂ to 458 ofSEQ ID NO:4, wherein x₂ is amino acid 1 or 17; amino acids x₃ to 439 ofSEQ ID NO:8, wherein x₃ is amino acid 1 or 17; amino acids x₄ to 458 ofSEQ ID NO:10, wherein x₄ is amino acid 1 or 17; amino acids x₅ to 242 ofSEQ ID NO:12, wherein x₅ is amino acid 1 or 17; amino acids x₆ to 461 ofSEQ ID NO:14, wherein x₆ is amino acid 1 or 17; amino acids x₇ to 449 ofSEQ ID NO:16, wherein x₇ is amino acid 1 or 17; amino acids x₈ to 259 ofSEQ ID NO:18, wherein x₈ is amino acid 1 or 17; amino acids x₉ to 443 ofSEQ ID NO:20, wherein x₉ is amino acid 1 or 17; and, amino acids x₁₀ to456 of SEQ ID NO:22, wherein x₁₀ is amino acid 1 or 17; b) a fragment ofany of the human P3G2 extracellular domains of a).
 13. An isolatedpolypeptide encoded by a DNA selected from the group consisting of: a)DNA capable of hybridizing under highly stringent conditions to DNAselected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6, thehighly stringent conditions including a hybridizing temperature of atleast 68° C.
 14. A fusion protein comprising amino acids 17 to 458 ofSEQ ID NO:2 and the Fc region of Ig.
 15. A fusion DNA constructcomprising DNA encoding amino acids 17 to 458 of SEQ ID NO:2 and DNAencoding the Fc region of Ig.
 16. A recombinant expression vectorcomprising DNA of claim
 1. 17. A process for preparing an LIRpolypeptide, the process comprising culturing a host cell transformedwith an expression vector of claim 16 under conditions that promoteexpression of said polypeptide, and recovering said polypeptide.
 18. Acomposition comprising a suitable diluent carrier and a polypeptide ofclaim
 8. 19. A host cell transformed or transfected with an expressionvector according to claim
 16. 20. An antibody that is immunoreactivewith a polypeptide of claim 8.